Systemic gene replacement therapy for treatment of X-linked MyoTubular Myopathy (XLMTM)

ABSTRACT

The present invention provides compositions and methods for treating a myopathy. In certain embodiments, the invention provides compositions and methods for treating, improving muscle function, and prolonging survival in a subject with X-linked myotubular myopathy (XLMTM). The present invention provides a method comprising systemic administration of a composition that induces the increased expression of myotubularin in the muscle of a subject. The invention provides sustained regional and global increases in muscle function.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/194,186, filed Feb. 28, 2014, allowed, which claims priorityunder 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/771,449,filed Mar. 1, 2013, each of which are hereby incorporated by referencein their entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P50 NS040828 andRO1 AR044345, awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Recent progress raises anticipation that in vivo gene replacement willsuccessfully treat many human genetic disorders (Epica, et al., 2002,Anim Genet 33(1):81-2; Guan et al., 2011, PM R 3(6 Suppl 1):S95-9;Forbes et al., 1985, AJR Am J Roentgenol 145(1):149-54). Nonetheless,substantial hurdles must be overcome to achieve regulatory approval forhuman gene therapies (Kornegay et al., 2011, Methods Mol Biol709:105-234), such as successful treatment in predictive animal models.X-linked myotubular myopathy (XLMTM; OMIM 310400) is a fatal monogenicdisease of skeletal muscle. Affected newborn boys, approximately one per50,000 births, typically display marked hypotonia and respiratoryfailure (Jungbluth et al., 2008, Orphanet J Rare Dis 3:26). Survivalbeyond the postnatal period requires intensive support, often includinggastrostomy feeding and mechanical ventilation (Herman et al., 1999, JPediatr 134(2):206-14). No effective therapy to treat this diseaseexists.

XLMTM results from loss-of-function mutations in Myotubularin 1 (MTM1)(Laporte et al., 1996, Nat Genet 13(2):175-82). This gene encodes one ofa family of 3-phosphoinositide phosphatases which act on the secondmessengers phosphatidylinositol 3-monophosphate [PI(3)P] andphosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] (Rosset et al., 2004,J Digit Imaging 17(3):205-16; Yue et al., 2011, Methods Mol Biol709:313-29; Salgado et al., 2003, JBR-BTR 86(4):215-20; Miyagoe-Suzukiand Takeda, 2010, Exp Cell Res 316(18):3087-92). Although myotubularinis expressed ubiquitously, loss of this enzyme primarily affectsskeletal muscle. Myogenesis occurs, but muscle fibers throughout thebody are hypotrophic and display structural abnormalities, withassociated weakness (Buj-Bello et al., 2002, Proc Natl Acad Sci USA99(23):15060-5).

The mammalian X-linked myotubularin gene is highly conserved (Laporte etal., 1998, Hum Mol Genet 7(11):1703-12; Laporte et al., 2000, Hum Mutat15(5):393-409; Beggs et al., 2010, Proc Natl Acad Sci USA107(33):14697-702). Genetic disruption of Mtm1 in mice causes profoundabnormalities in skeletal muscle mass, structure, and function,regardless whether expression is knocked out constitutively or only inmuscle (Buj-Bello et al., 2002, Proc Natl Acad Sci USA 99(23):15060-5;Al-Qusairi et al., 2009, Proc Natl Acad Sci USA 106(44):18763-8). Thephenotype resembles human XLMTM, with similar pathology and earlymortality.

Thus there is a need in the art for effective and non-invasivecompositions and methods for treating myopathy, including XLMTM. Thepresent invention satisfies this unmet need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a method of treating a myopathy in a subject inneed thereof, the method comprising systemically administering to thesubject a composition that increases the expression of myotubularin in amuscle of the subject.

The invention further includes a method of prolonging the survival of asubject with myopathy, the method comprising systemically administeringto the subject a composition that increases the expression ofmyotubularin in a muscle of the subject.

The invention also includes a composition comprising a nucleic acidsequence comprising MTM1, wherein the composition is suitable forsystemic delivery to a subject, further wherein the compositioncomprises a viral vector.

In certain embodiments, the composition comprises a nucleic acidsequence encoding myotubularin. In other embodiments, the compositioncomprises the myotubularin gene (MTM1). In yet other embodiments, thecomposition further comprises an expression vector comprising a viralvector. In yet other embodiments, the composition further comprises amuscle specific promoter.

In certain embodiments, the viral vector is selected from the groupconsisting of a lentiviral vector, retroviral vector, adenoviral vector,and adeno-associated viral (AAV) vector.

In other embodiments, the AAV vector comprises a serotype selected fromthe group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,and AAV9. In yet other embodiments, the AAV vector is a recombinant AAVvector. In yet other embodiments, the composition comprisesmyotubularin, or a biologically functional fragment thereof.

In certain embodiments, the myopathy comprises X-linked myotubularmyopathy (XLMTM). In other embodiments, the administration routecomprises at least one selected from the group consisting of enteral,parenteral, oral, intravenous, intra-arterial, and inhalational. In yetother embodiments, the administration route comprises intravenous.

In certain embodiments, the muscle of the subject exhibits an increasein myotubularin expression for up to 6 months as compared to the muscleof the subject in the absence of administration of the composition. Inother embodiments, the muscle of the subject exhibits an increase inmyotubularin expression for up to 1 year as compared to the muscle ofthe subject in the absence of administration of the composition. In yetother embodiments, the muscle of the subject exhibits a sustainedincrease in strength for up to 6 months as compared to the muscle of thesubject in the absence of administration of the composition. In yetother embodiments, the subject has longer survival than a subject who isnot administered the composition. In yet other embodiments, the functionof the diaphragm of the subject is improved as compared to the diaphragmof the subject in the absence of administration of the composition.

In certain embodiments, a single administration of the composition isperformed in the subject within a period of time. In other embodiments,two or more administrations of the composition are performed in thesubject within a period of time. In yet other embodiments, the subjectis a mammal. In yet other embodiments, the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1E depict the results of experiments demonstrating thatintravascular delivery of rAAV-Mtm1 in myotubularin-deficient miceimproves lifespan and body growth. (FIG. 1A) Experimental design. (FIG.1B) Survival and (FIG. 1C) body mass of wild-type mice (WT) andconstitutive KO-Mtm1 mice injected at 3 weeks of age with saline(WT+saline, KO+saline, n=10 per genotype). Myotubularin-deficient micewere injected with rAAV-Mtm1 at 3×10¹³ viral genomes per kg (vg/kg) at 3(KO+AAV, n=12) and 5 weeks of age (KO Late+AAV, n=12) during a 6 monthsfollow-up study. (FIG. 1D) Mass of representative skeletal muscles ofKO-Mtm1 mice 2 weeks after injection of saline (KO+saline, n=4) and 6months (n=10 after injection of rAAV-Mtm1 (KO+AAV, n=7, and KO Late+AAV,n=8). Values were normalized to muscle mass of age-matched,saline-injected WT mice (n=10), taken as 100%. (FIG. 1E) Myotubularinprotein quantification by immunoblot compared to endogenous levels(line=1); GAPDH immunodetection was used an internal control. The numberof animals was as in FIG. 1D. Muscles: TA=tibialis anterior;EDL=extensor digitorum longus; SOL=soleus; GA=gastrocnemius;QUA=quadriceps; TRI=triceps; BI=biceps brachii; DIA =diaphragm.Statistical significance: P<0.05 (one symbol); P<0.01 (two symbols);P<0.001 (three symbols); Mann-Whitney test, each condition versusWT+saline values.

FIGS. 2A-2B depict the results of experiments demonstrating that Mtm1gene replacement therapy corrects the internal architecture andhypotrophy of skeletal muscle fibers in myotubularin-knockout mice.Treatment groups were as described in FIG. 18. Mice were injected witheither saline (+saline) or rAAV-Mtm1 vector (+AAV). Sections wereobtained after 2 weeks (5 weeks of age) and 6 months of treatment. (FIG.2A) Cross-sections from tibialis anterior (TA) muscle stained withhematoxylin and eosin (HE) and NADH-TR, and by immunofluorescence withantibodies against DHPR1α and dysferlin. Scale bars=10 μm. (FIG. 2B)Mean diameter of muscle fibers from TA and biceps brachii muscles frommice injected with either saline or rAAV-Mtm1 after 2 weeks (left graph)and 6 months of treatment (right graph). WT+saline, n=10; KO+saline,n=4; KO+AAV, n=7, and KO Late+AAV, n=8. Statistical significance: P<0.05(one symbol, *); P<0.01 (two symbols, **); P<0.001 (three symbols, ***);t test, each condition versus WT+saline values.

FIGS. 3A-3C depict the results of experiments demonstrating that genereplacement therapy with rAAV8-Mtm1 improves strength, activity andlong-term survival in myotubularin deficient mice. (FIG. 3A) Whole-bodyspontaneous mobility of normal (WT+saline), mutant (KO+saline) andAAV-treated mutant (KO+AAV-Mtm1) mice 2 weeks (5 weeks of age) and 6months after PBS or vector injection. The distance covered over the90-min test was assessed using an open field actimeter. (FIG. 3B) Escapetest measurements in the 5 groups of mice. (FIG. 3C) Specific tetanicforce of isolated EDL muscles from KO mice injected at an early and latestage of the disease 6 months after vector delivery compared tosaline-injected KO and WT littermates. Drawings of the tests are shownon the left side of each figure section. WT+saline, n=6; KO+saline, n=4at 5 weeks; WT+saline, n=10; KO+saline, n=4; KO early+AAV, n=8, and KOLate+AAV, n=8 at 6 months. Statistical significance: P<0.05 (one symbol,*); P<0.01 (two symbols, **); P<0.001 (three symbols, ***); t test.

FIGS. 4A-4C depict the results of experiments demonstrating thatmyotubularin is expressed and increases muscle mass and volume afterlocal gene therapy in XLMTM dogs. (FIG. 4A) Cranial tibialis caninemuscles 6 weeks after AAV8-MTM1 intramuscular injection. Immunoblot ofmyotubularin (MTM1) and GAPDH from cranialtibialis muscle lysates atproximal, middle, or distal sections of the muscle. (FIG. 4B) MTM1 genereplacement therapy increases hindlimb strength in XLMTM dogs. Thedrawing shows the method used to measure hindlimb flexion strength indogs. A nerve stimulator delivers electrical frequencies from 1 to 110Hz to muscles that pull the paw toward the stifle (knee). A transducercaptures the torque generated when the paw pulls on the foot pedal.Upper graph: baseline before injection, 10 weeks of age (WT, n=3; XLMTM,n=3); middle graph: 4 weeks after injection, 14 weeks of age (WT, n=3;XLMTM, n=3); bottom graph: 6 weeks after injection, 16 weeks of age (WT,n=2; XLMTM, n=2). P=0.002 (4 weeks after injection); P=0.0161 (6 weeksafter injection; XLMTM+AAV versus WT+saline values), one-way analysis ofvariance (ANOVA). (FIG. 4C) Local myotubularin gene replacement therapyimproves muscle fiber architecture in XLMTM dogs. Cryosections of thecranial tibialis muscle (middle part) were assessed microscopically:Grey boxes in NADH-TR staining show areas magnified below;immunofluorescence staining for DHPR1α and dysferlin shows correction ofabnormal organelles with AAV8-MTM1 (large grey arrow). Scale bars, 25mm. Electron microscopy (EM) shows normal T-tubules (small black arrows)and abnormal L-tubules (small white arrow). Scale bar, 500 nm. Bottompanel: Close-up of WT muscle and schematic showing normal relationshipof sarcomere ends (Z-line), and triads of T-tubules and sarcoplasmicreticulum (SR).

FIGS. 5A-5G depict the results of experiments demonstrating increase inhindlimb strength of XLMTM dogs after intravascular administration ofAAV8-MTM1. Data are presented as means±standard deviation (SD) combinedvalues of both limbs. (FIG. 5A) Peak hindlimb torque at various times upto 1 year after infusion. (FIG. 5B) Baseline before infusion, 9 weeks ofage (WT, n=2; XLMTM+AAV8, n=3; XLMTM+saline, n=1). (FIG. 5C) Six weeksafter infusion, 15 weeks of age (WT, n=2; XLMTM+AAV8, n=2; XLMTM saline,n=2). (FIG. 5D) Eight weeks after infusion, 17 weeks of age (WT, n=3;XLMTM+AAV8, n=3; XLMTM+saline, n=3). (FIG. 5E) Fourteen weeks afterinfusion, 23 weeks of age (WT, n=3; XLMTM+AAV8, n=3). XLMTM dogs infusedonly with saline did not survive beyond 18 weeks of age. (FIG. 5F) Oneyear after infusion (WT, n=1; carrier, n=3; XLMTM+AAV8, n=3). (FIG. 5G)Pic inspiratory flow (PIF), a respiratory functional measure reflectingdiaphragm muscle strength, taken in anesthetized dogs at baseline and at8 weeks, 14 weeks, and 1 year after infusion with AAV8. Number ofanimals per group was the same as in (FIG. 5B) to (FIG. 5F). P<0.001(three symbols, ***); one-way ANOVA, each condition versus WT+salinevalues.

FIGS. 6A-6B depict the results of experiments demonstrating that MTM1gene therapy corrects the internal architecture and hypotrophy ofskeletal muscle fibers in myotubularin-mutant dogs. Muscle cryosectionsfrom age-matched WT or AAV8-MTM1-infused XLMTM dogs were assessedmicroscopically. Comparison is shown between the left (infused) hindlimband the right (contralateral noninfused) limb. (FIG. 6A) Representativemicrographs of quadriceps muscle cross sections from muscle biopsiestaken 4 weeks after infusion from dog 4 stained with H&E or NADH-TR.(FIG. 6B) H&E-stained cranial tibialis muscle cross sections taken 1year after AAV infusion. Graphs indicate myofiber diameter frequencydistribution of corresponding images for the vastus lateralis muscle(upper graph) and craniotibialis muscle (lower graph). Scale bars, 25mm.

FIGS. 7A-7D depict the results of experiments demonstrating thebiodistribution of AAV8 vector and the myotubularin transgene expressionin XLMTM dogs infused with AAV8-MTM1. (FIGS. 7A-7B) Comparison of vectordistribution (FIG. 7A) and MTM1 transgene expression (FIG. 7B) amongupper and lower limb muscle biopsies collected 4 weeks after infusion inthree XLMTM dogs: dogs 4 to 6. Upper limb muscles: triceps (TRI) andbiceps brachii (BI bra). Lower limb muscles: biceps femoris (BI fem) andquadriceps (QUA). R, right; L, left; inf, infused limb. (FIG. 7B)Expression of canine MTM1 protein relative to the housekeeping geneGAPDH in whole-muscle lysates probed with anti-myotubularin antibody.(FIGS. 7C-7D) Comparison of vector distribution (FIG. 7C) and MTM1transgene expression (FIG. 7D) among upper and lower limb musclenecropsy samples collected 1 year after infusion in dog 4, an XLMTM dog.Muscles of the infused leg: VL, vastus lateralis; VM, vastusmedialis;RF, rectus femoris; Ad, adductor magnus; Pec, pectineus; Sar, cranialsartorius; Gra, gracilis; BI fem, biceps femoris; ST, semitendinosus;SM, semimembranosus; CT, cranial tibialis; Gast, gastrocnemius; Per,peroneus longus. Muscles distal to the infused leg: Dia, diaphragm;Interc, intercostals; He, heart.

FIGS. 8A-8B depict the results of experiments demonstrating thepathology of muscles from 3 weeks-of-age myotubularin deficient(KO-Mtm1) mice. (FIG. 8A) Left panel: Body mass of wild-type (WT) andKO-Mtm1 mice before injection. Right panel mass of representativeskeletal muscles: quadriceps (QUA), tibialis anterior (TA), triceps(TRI) (n=9 for each genotype, both contralateral muscles were analyzedper mouse). Note the significant reduction of TA muscle mass in KO-Mtm1mice (P<0.01). (FIG. 8B) Hematoxylin and eosin (H&E) and nicotinamideadenine dinucleotide tetrazolium reductase (NADH-TR) staining of TAmuscle cross-sections. Note the presence of small myofibers,internalized nuclei and altered mitochondrial oxidative stainingdistribution in KO-Mtm1 muscle. Scale bars=50 μm.

FIGS. 9A-9C depict the results of experiments demonstrating thatsystemic gene replacement therapy ameliorates pathological hallmarks ofmyotubular myopathy in skeletal muscles. Constitutive Mtm1 knockout mice(KO-Mtm1) at 3 weeks of age received a single intravenous injection ofrAAV-Mtm1 as described elsewhere herein (see FIG. 1). Saline injectedKO-Mtm1 and WT mice served as controls. (FIG. 9A) Distribution ofmyofiber diameters of tibialis anterior (TA, upper right panels) andbiceps brachii (BI, lower right panels) muscles at 6 monthspost-injection in the two group of Mtm1 KO animals (3 weeks and 5 weeksof age at injection). Left panels show the distribution of fibers from 5weeks old WT and KO mice (FIG. 9B) Nuclei internalization. Thepercentage of myofibers with internal nuclei was quantified in tibialisanterior muscle and biceps brachii of mice from the various treatmentconditions. (FIG. 9C) Myotubularin protein quantification in heart byimmunoblot 6 months after vector administration; GAPDH immunodetectionwas used as an internal control (WT+saline, n=10 and KO Early+AAV, n=7),Statistical significance: P<0.05 (one symbol, *); P<0.01 (two symbols,**); P<0.001 (three symbols, ***); t-test, each condition versusWT+saline. Number of animals: WT+saline, n=6 and KO+saline, n=4 at 5weeks; WT+saline, n=10, n=4 KO Early+AAV, n=8, and KO Late+AAV, n=8 at 6months.

FIGS. 10A-10E depict the results of experiments demonstrating thatintravascular delivery of a lower dose of AAV8-Mtm1 inmyotubularin-deficient mice improves partially life span and bodygrowth. (FIG. 10A) Survival and (FIG. 10B) body mass of wild type mice(WT) and constitutive KO-Mtm1 mice during a 3 months follow-up study.Mice received either saline (WT+saline, n=20, KO+saline, n=10) or theAAV8-Mtm1 vector at 3×10¹³ vg/mL (KO+AAV, n=10) and 5×10¹² vg/mL (KO+AAVLow, n=10). (FIG. 10C) Mass of representative skeletal muscles of Mtm1KO mice 3 months after vector injection. Values were normalized tomuscle mass of age-matched, saline-injected WT mice, taken as 100%.Muscles: TA=tibialis anterior; EDL=extensor digitorum longus;SOL=soleus; GA=gastrocnemius; QUA=quadriceps; TRI=triceps; BI=bicepsbrachii. (FIG. 10D) Myotubularin protein quantification by immunoblotcompared to endogenous levels (line=1); GAPDH immunodetection was usedan internal control. (FIG. 10E) Distance covered in the 90-min actimetertest, global strength in the escape test and specific tetanic force ofisolated EDL and soleus muscles of mice 3 months post-injection. In(FIG. 10C) and (FIG. 10D), n=7 for KO+AAV, n=5 for KO+AAV low and n=10for WT+saline). Statistical significance: P<0.05 (one symbol, *); P<0.01(two symbols, **); P<0.001 (three symbols, ***); each condition versusWT+saline values.

FIGS. 11A-11B depict the results of experiments demonstrating thattargeted myotubularin gene replacement therapy in XLMTM dogs increasesthe overall size of injected muscles. (FIG. 11A) Necropsy photos (toppanel) of the injected canine hind limb muscles in comparison to CTreconstruction images (bottom panel). In each pair the left image isfrom the limb injected with rAAV-MTM1, and the right image is thesaline-treated control (see also FIG. 4). (FIG. 11B) Muscle volume ofinjected hind limbs muscles 1 day prior to necropsy. XLM™=affected dog;WT=unaffected littermate; +MTM1=injected with rAAV-MTM1 at 10 weeks ofage (4 or 6 weeks prior to necropsy). Scale bars: 2 cm.

FIGS. 12A-12D depict the results of experiments demonstrating thattargeted gene therapy with rAAV-MTM1 injected into the cranial tibialismuscle improves in vivo contractile response to repeated lengthening(eccentric) contractions in XLMTM dogs. (FIG. 12A) Anesthetized dogs arepositioned to allow the hind foot to rotate around the axis of a forcetransducer during repeated contractions. (FIGS. 12B-12D) Isometricflexion torque values are shown immediately following each of 30eccentric contractions. Measurements were obtained (FIG. 12B) atbaseline, 10 weeks-of-age, prior to injection (XLMTM n=3); (FIG. 12C) 4weeks post-injection (XLMTM n=3); (FIG. 12D) 6 weeks post-injection(XLMTM n=2). WT=unaffected littermates (n=3). Two-way ANOVA was used tocompare saline-treated XLMTM versus vector-treated XLMTM.

FIGS. 13A-13C depict the results of experiments demonstrating the invivo strength measured 1 year after regional hindlimb infusion ofAAV8-MTM1 in an XLMTM dog. (FIG. 13A) Hindlimb flexion strength incontralateral non-infused versus infused muscles of Dog 4, assessed 1year after infusion. (FIG. 13B) CT reconstruction and (FIG. 13C) crosssection through the thigh demonstrating marked hypertrophy of theinfused limb (marked by a symbol *).

FIG. 14 represents the results of a western blot of myotubularintransgene expression in XLMTM dogs infused with AAV8-MTM1. Theexpression of canine MTM1 protein is shown relative to the housekeepinggene, GAPDH in whole muscle lysates probed with an anti-myotubularinantibody (see FIGS. 7A-D for expression levels in various muscles oneyear post-infusion). XLMTM muscle infused with saline only: lanes 1-2;XLMTM infused with AAV8-MTM1: lanes 3-4; and wild type muscles withoutinfusion: lanes 5-8.

FIG. 15 displays the necropsy findings in the heart of an XLMTM dog 1year following AAV8-MTM1 infusion. Pathological assessment of the heartof D4 at the gross (left panel) and microscopic (right panel) levels didnot reveal histopathological or structural abnormalities. Legend:RV=right ventricle; LV=left ventricle; Mason=mason's trichrome stain.Size bars=40 μm.

FIGS. 16A-16D depict the results of experiments demonstrating thehumoral response specific to AAV8 and myotubularin in XLMTM dogs. (FIGS.16A-16B) Serum neutralizing factor (NAF), IgM and IgG antibodies againstthe AAV8 capsid. Sera were analyzed before and after intramuscular (FIG.16A) or regional limb (FIG. 16B) administration of AAV8-MTM1. (FIGS.16C-16D) Humoral response specific to MTM1 protein in XLMTM dogs. Serawere analyzed before and after intramuscular (FIG. 16C) or regional limb(FIG. 16D) administration of AAV8-MTM1.

FIGS. 17A-17B depict the results of experiments demonstrating thecellular response to rAAV8 (FIG. 17A) or MTM1 protein (FIG. 17B) inXLMTM dogs. Values indicate the LV-VP1:LV-empty ratios. Results areexpressed as spot-forming units/10⁶ cells. Samples were consideredpositive for the antigen if the number of spots was greater than 1.5times the corresponding control with LV-empty. Assays were scored if thenumber of spots under stimulation >15 spots per 2×10⁵ PBMC. NA=Notapplicable

FIG. 18 depicts the results of experiments demonstrating the cellularresponse to AAV8 or MTM1 protein in XLMTM dogs. Results are expressed asratio of number of spot-forming units secreting IFN-γ per 10⁶ PBMC afterstimulation with LV-Cap8 or LV-MTM1 to number of spot-forming unitssecreting IFN-γ per 10⁶ PBMC after stimulation with LV-empty, for eachdogs. Nd indicates not done. Samples were considered positive for theantigen if the number of spots was greater than 1.8 timesthecorresponding control with LV-empty. Assays were scored if the numberof spots under stimulation >15 spots per 2×10⁵ PBMC. NA=Not analyzed.

FIGS. 19A-19H depict the results of experiments demonstrating thatintravascular delivery of rAAV-Mtm1 in myotubularin-deficient miceimproves lifespan and body growth. (FIG. 19A) Survival of wild-typeC57BL/6 mice (WT) injected at 3 weeks-of-age with saline (WT+saline,n=5) and constitutive KO-Mtm1 mice, of same age, injected with saline(KO+saline, n=10) or with rAAV-Mtm1 at 3.0×10¹³ viral genomes per kg(vg/kg) (KO+AAV-Mtm1, n=5) during a 6 months follow-up study. (FIG. 19B)Body mass of saline-injected WT (n=11) and KO-Mtm1 (n=16) andrAAV9-Mtm1-injected KO (n=8) mice. (FIG. 19C) Mass of representativeskeletal muscles of KO-Mtm1 mice 2 weeks after injection of saline(KO+saline, n=8) and 2 weeks (n=6) or 6 months (n=10) after injection ofrAAV-Mtm1 (KO+AAV-Mtm1). Values were normalized to muscle mass ofage-matched, saline-injected WT mice (n=12), taken as 100%. (FIG. 19Dand FIG. 19H) Myotubularin protein quantification by immunoblot. Insetsshow representative blots for myotubularin (MTM1) and glyceraldehyde3-phosphate dehydrogenase (GAPDH), used for normalization. (FIG. 19E)Survival of WT mice after injection at 4 weeks-of-age with saline(WT+saline, n=8) and of muscle-specific mKO-Mtm1 knockout mice afterinjection at same age with saline (mK0+saline, n=5) or rAAV-Mtm1 at0.5×10¹³ vg/kg (mK0+AAV-Mtm1, n=6) during a 12 months follow-up study.(FIG. 19F) Body weights of mice from panel e. (FIG. 19G) Masses ofrepresentative skeletal muscles of mKO-Mtm1 mice at 12 months aftertreatment with rAAV-Mtm1, normalized to muscle masses of controlWT+saline mice, taken as 100%. Muscles: TA=tibialis anterior;EDL=extensor digitorum longus; GA=gastrocnemius; QUA=quadriceps;TRI=triceps; BI=biceps brachii; DIA=diaphragm. Statistical significance:P<0.05 (one symbol, *); P<0.01 (two symbols, **). Symbols: * (WT+saline)versus (KO+saline); A (KO+saline) versus (KO+AAV-Mtm1); # (WT+saline)versus (KO+AAV-Mtm1).

FIGS. 20A-20F depict the results of experiments demonstrating that Mtm1gene replacement therapy corrects the internal architecture andhypotrophy of skeletal muscle fibers in myotubularin-knockout mice.Treatment groups were as described in FIGS. 1A-1B. WT=C57BL/6 mice;KO=constitutive KO-Mtm1 mice. Mice were injected with either saline(+saline) or rAAV vector (+Mtm1). Sections were obtained at 2 weeks and6 months. (FIG. 20A) Whole muscle cross-sections from tibialis anterior(TA) stained with hematoxylin and eosin (H&E). Histological staining ofmuscle: (FIG. 20B) H&E, (FIG. 20C) NADH-TR. Immunofluorescence (IF):(FIG. 20D) DHPR1α, (FIG. 20E), dysferlin. Scale bars=10 μm. (FIG. 20F)Size distribution of TA myofibers by morphometry.

FIGS. 21A-21D depict the results of experiments demonstrating thatMyotubularin is expressed and increases muscle mass and volume afterlocal gene therapy in XLMTM dogs. (FIG. 21A) Immunoblot of myotubularin(MTM1, green) and GAPDH (red) from cranial tibialis muscle lysatesfollowing injection of XLMTM and unaffected (WT) dogs with either salineor rAAV-MTM1 vector. Lanes 2 and 3, 4 and 5, 6 and 7 show results fromcontrol (saline) and vector-treated contralateral limbs, respectively,of three XLMTM dogs. Lane 1: (WT+saline). Lanes 2, 4, 6: (XLMTM saline).Lanes 3, 5, 7: (XLMTM+rAAV-MTM1). (FIG. 21B) Relative levels byquantitative immunoblotting of myotubularin (normalized to GAPDH) incranial tibialis muscle after saline injection in unaffected dog (WT),and after vector (4×10¹¹ vg) injection in XLMTM dogs. Muscles samplesobtained at necropsy (6 weeks), n=2. Samples from vector-treated XLMTMdogs were taken from near the injection site at the center of thecranial tibialis muscle (black) or pooled from the distal and proximalends of that muscle (grey). The myotubularin level was approximately 60%of WT at the muscle center, and approximately 8% of WT at the ends.

(FIG. 21C) Computed tomography (CT) reconstruction at 6 weekspost-injection of treated (+AAV-MTM1) and control (saline) hind limbs ofan XLMTM dog. (FIG. 21D) Cranial tibialis muscle mass. Symbols forstatistical significance: same as in FIG. 19A-19H.

FIGS. 22A-22B depict the results of experiments demonstrating thatsystemic gene replacement therapy ameliorates hypotrophy of skeletalmuscles throughout the body of myotubularin-deficient mice. ConstitutiveMtm1 knockout mice (KO-Mtm1) at 3 weeks-of-age received a singleintravenous injection of rAAV-Mtm1. Saline injected KO-Mtm1 and WT miceserved as controls. (FIG. 22A) Mean myofiber diameters of individualskeletal muscles at 2 weeks and 6 months post-injection. Muscles:TA=tibialis anterior; EDL=extensor digitorum longus; SOL=soleus;TRI=triceps; BI=biceps brachii. (FIG. 22B) Distribution of myofiberdiameters of BI muscles at 2 weeks and 6 months post-injection. Numbersof animals: at 2 weeks after injection (WT+saline, n=6), (KO+saline,n=4), (KO+Mtm1, n=3); at 6 months after injection (WT+saline, n=5),(KO+Mtm1, n=5). Symbols for statistics as in FIG. 19.

FIGS. 23A-23C depict the results of experiments demonstrating thecorrection of structural and functional muscle defects by Mtm1 genereplacement therapy in muscle-specific myotubularin deficient mice. Asingle intravenous injection of rAAV-Mtm1 (0.5×10¹³ vg/kg) wasadministered to mKO-Mtm1 mice at 4 weeks-of-age, as described inMethods. Age-matched, saline-injected mKO-Mtm1 and wild-type C57BL/6(WT) mice served as controls. (FIG. 23A) Nuclei internalization. Thepercentage of myofibers with internal nuclei was quantified in tibialisanterior muscle. (FIG. 23B) Whole-field actimeter determination ofdistance traveled in 90 minutes, determined at 12 months. The distancecovered by treated mutant mice (mKO+AAV-Mtm1) was not significantlydifferent from that of WT littermates. (FIG. 23C) Specific tetanic forceof isolated EDL muscles 12 months after vector delivery. P<0.01 (twosymbols, ##). In all cases n=7 for (WT+saline), n=5 for (mKO+AAV-Mtm1).

FIG. 24 depicts the results of experiments demonstrating thatMyotubularin gene therapy restores normal muscle ultrastructure inmyotubularin-deficient mice. Electron micrographs of TA muscles at 6months after injection of WT mice with saline and of KO-Mtm1 mice withrAAV-Mtm1. Both groups show similar numbers and structure of T-tubules(arrows) and triads (arrowheads). Scale bars: 1 μm (upper panels); 500nm (lower panels).

FIGS. 25A-25B depict the results of experiments demonstrating thatMyotubularin is expressed following intravenous limb infusion ofrAAV8-MTM1 in XLMTM dogs. (FIG. 25A) AAV8 vector copy number perchromosome expressed in various muscles from infused XLMTM dogs. (FIG.25B) Immunoblot of myotubularin (MTM1) and GAPDH from muscle biopsylysates 4 weeks after rAAV8-MTM1 infusion in XLMTM dogs. Lanes 1 and 8were loaded with normal (WT) muscle lysates, lanes 2 and 6 fromuntreated XLMTM muscle lysates, and the remaining lanes loaded withlysates from XLMTM dogs infused with rAAV8-MTM1, with the location ofthe muscle samples taken from areas indicated by the colored arrows.Muscles samples were obtained at biopsy (4 weeks after infusion)

FIGS. 26A-26C depict the results of experiments demonstrating thatintravenous myotubularin gene replacement therapy improves muscle fiberarchitecture in XLMTM dogs. Muscle cryosections from age-matchedwild-type (WT) or rAAV8-MTM1 infused XLMTM dogs were assessedmicroscopically. Comparison is shown between the left (infused) hindlimband the right (contralateral non-infused) limb. (FIG. 26A) Hematoxylinand eosin (H&E) and (FIG. 26B) nicotinamide adenine dinucleotidetetrazolium reductase (NADH-TR) staining of muscle cross-sections. Sizebars=25 μm. (FIG. 26C) Electron microscopy (EM) shows similar numbersand structure of T-tubules (arrows) and triads (arrowheads) Bar=500 nm.

FIGS. 27A-27B are a set of images depicting the use of RespiratoryImpedance Plethysmography (RIP) to measure diaphragm strength. FIG. 27Adepicts the abdominal and thoracic bands worn by the subject. FIG. 27Bdepicts the exemplary output from the bands.

FIG. 28 is a graph depicting the results of experiments demonstratingthat systemic AAV8-MTM1 delivery improves movement of diaphragm musclein XLMTM subjects.

FIG. 29 is a schematic of flow versus time as measured by a pneumotach.The area under the curve is used to calculate volume

FIG. 30 is a graph depicting the results of experiments demonstratingthat the TBFVL of XLMTM subjects treated with systemic AAV8-MTM1 isimproved over untreated subjects, thereby revealing improved diaphragmfunction.

FIG. 31 is a graph depicting the TBFVL and the peak inspiratory flow ofwild type and XLMTM dogs.

FIG. 32 is a graph depicting the results of experiments demonstratingthat peak inspiratory flow improves flowing systemic delivery ofAAV8-MTM1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for treatment ofmyopathy in a subject in need thereof. The present invention relates toa strategy of delivering the myotubularin gene (MTM1) to subjects inneed of improved muscle function. The compositions and methods of thepresent invention increase the formation of muscle and improve musclefunction in the subject.

In one embodiment, the present invention is useful for treating anindividual with a myopathy. In one embodiment, the present invention isuseful for treating an individual with X-linked myotubular myopathy(XLMTM). XLMTM is a fatal pediatric disease of skeletal muscle,characterized by a loss of function mutation in MTM1. The presentinvention improves muscle function and prolongs survival in afflictedsubjects. However, the present invention is not limited to subjectshaving XLMTM. Rather, the present invention is applicable to improvingmuscle function in any subject in need of improved muscle function.

In one embodiment, the present invention provides a composition thatincreases the expression of myotubularin in a subject. In one embodimentthe composition comprises a nucleic acid comprising a nucleic acidsequence encoding myotubularin. In another embodiment, the compositioncomprises MTM1. In yet another embodiment, the composition comprises aviral vector comprising a nucleic acid sequence encoding myotubularin.In yet another embodiment, the composition further comprises a mechanismfor specific translation of MTM1 within muscle tissue.

The present invention relates to the findings that systemic delivery ofan adeno-associated viral vector encoding MTM1 drastically improvesmuscle function and provides long-term prolonged survival. Thus, thepresent invention provides non-invasive compositions and methods totreat a myopathy, including XLMTM.

In one embodiment, the present invention provides a method for improvingmuscle function comprising administering an effective amount of acomposition which increases myotubularin expression in a subject. In oneembodiment, the method comprises administering to a subject acomposition comprising a nucleic acid comprising a nucleic acid sequenceencoding myotubularin. In another embodiment, the composition comprisesMTM1. In yet another embodiment, the method comprises injection of acomposition comprising MTM1 directly into a muscle of a subject in needof improved muscle function. In another embodiment, the method comprisessystemic delivery of a composition comprising MTM1 to a subject in needof improved muscle function. In one embodiment, the method provides asustained elevated level of myotubularin in a subject, which leads tosustained improvements in muscle strength, size, and function.

In another embodiment, the present invention provides a method forprolonging the survival of subjects having a myopathy. The methodcomprises administering an effective amount of a composition comprisingMTM1 to a subject having a myopathy. For example, in one embodiment, themethod comprises administering a nucleic acid comprising a nucleic acidsequence encoding myotubularin. In another embodiment, the methodcomprises injection of a composition comprising MTM1 directly into themuscle of the subject. In another embodiment, the method comprisessystemic delivery of a composition comprising MTM1 to the subject. Inyet another embodiment, the method provides a sustained elevated levelof myotubularin in the subject, which leads to prolonged survival. In afurther embodiment, the subject having a myopathy has XLMTM.

In certain embodiments, systemic delivery comprises delivery of thecomposition to the subject such that composition is accessiblethroughout the body of the subject. For example, in certain embodiments,systemic delivery comprises enteral, parenteral, oral, intravenous,intra-arterial, and/or inhalational administration. In otherembodiments, systemic delivery of the composition comprisesadministering the composition near a local treatment site (i.e. in avein or artery nearby a weakened muscle). The present invention relatesto the discovery that intravenous administration of a vector comprisingMTM1 strengthened muscle regionally and globally. Thus, in certainembodiments, the invention comprises a local delivery of the compositionwhich produces systemic effects. In one aspect, systemic deliveryinduces improved muscle strength and function in the diaphragm, whosefunction is critical for quality of life and survival. In certainembodiments, systemic delivery is preferred as local delivery to thediaphragm is invasive and comes with great risk of damaging internalorgans.

In certain embodiments, the method comprises a single systemic deliveryof a composition comprising MTM1. As described herein, the presentinvention is partly based upon the discovery that a single systemicdelivery of a vector comprising MTM1 produced sustained increases inmyotubularin expression and muscle function. In certain embodiments, themethod induces increased expression of myotubularin in a subject for aperiod of time, such as 1 day, 3 days, 1 week, 2 weeks, 1 month, 3months, 6 months, 1 year, 5 years, or longer. In other embodiments, themethod comprises two, three or more systemic deliveries of a compositioncomprising MTM1, wherein the administrations take place within a periodof time, such as 1 day, 3 days, 1 week, 2 weeks, 1 month, 3 months, 6months, 1 year, 5 years, any fraction or combination of time therein, orlonger.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting there from. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

“Parenteral” administration of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, “treating a disease or disorder” means reducing thefrequency with which a symptom of the disease or disorder is experiencedby a patient. Disease and disorder are used interchangeably herein.

The phrase “therapeutically effective amount,” as used herein, refers toan amount that is sufficient or effective to prevent or treat (delay orprevent the onset of, prevent the progression of, inhibit, decrease orreverse) a disease or condition, including alleviating symptoms of suchdiseases.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention provides compositions and methods for improvingmuscle function. In one embodiment, the present invention provides for atreatment of a myopathy. The myopathy may be any form of myopathy,including inherited or acquired myopathies. In one embodiment, thepresent invention provides compositions and methods for treatingX-linked myotubular myopathy (XLMTM). However, the present invention isnot limited to treatment of any particular disorder(s). Rather, theinvention provides for the ability to improve muscle function in anysubject in need of improved muscle function. For example, in oneembodiment, the present invention improves muscle function in subjectswhose muscles have weakened or atrophied.

The present invention relates to the findings in higher order animalmodels of XLMTM that systemic delivery of a viral vector comprising theMTM1 gene drastically improves regional and global muscle function andresults in prolonged survival. Thus, the compositions and methodsdescribed herein are useful in that they provide an easy and efficienttreatment of muscular disorders, including XLMTM.

Compositions

The present invention provides a composition that increases theexpression of myotubularin, or a biologically fragment thereof, in amuscle. For example, in one embodiment, the composition comprises anisolated nucleic acid sequence comprising MTM1, or a biologicallyfunctional fragment thereof. The MTM1 gene encodes myotubularin, amuscle specific protein integral for muscle function. As describedherein, delivery of a composition comprising MTM1 improves musclefunction. Furthermore, the delivery of a composition comprising MTM1prolongs survival of a subject having a loss of function mutation inMTM1.

In one embodiment, the composition comprises an isolated nucleic acidcomprising a sequence encoding myotubularin, or a biologicallyfunctional fragment thereof. In one embodiment, the nucleic acidcomprises a sequence comprising at least one of SEQ ID NOs: 1-6. Theisolated nucleic acid sequence encoding myotubularin can be obtainedusing any of the many recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned. In one embodiment, the composition

In certain embodiments, the composition increases the expression of abiologically functional fragment of myotubularin. For example, in oneembodiment, the composition comprises an isolated nucleic acid sequenceencoding a biologically functional fragment of myotubularin. In anotherembodiment, the composition comprises a biologically functional fragmentof MTM1. As would be understood in the art, a biologically functionalfragment is a portion or portions of a full length sequence that retainthe biological function of the full length sequence. Thus, abiologically functional fragment of myotubularin comprises a peptidethat retains the function of full length myotubularin. Further, abiologically functional fragment of MTM1 comprises a nucleic acidsequence which encodes myotubularin, or biologically functional fragmentthereof.

Further, the invention encompasses an isolated nucleic acid encoding apeptide having substantial homology to the peptides disclosed herein.Preferably, the nucleotide sequence of an isolated nucleic acid encodinga peptide of the invention is “substantially homologous”, that is, isabout 60% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous, more preferably about 90% homologous,even more preferably, about 95% homologous, and even more preferablyabout 99% homologous to a nucleotide sequence of an isolated nucleicacid encoding a peptide of the invention.

The present invention also includes a vector in which the isolatednucleic acid of the present invention is inserted. The art is repletewith suitable vectors that are useful in the present invention.

In brief summary, the expression of natural or synthetic nucleic acidsencoding myotubularin is typically achieved by operably linking anucleic acid encoding the myotubularin or portions thereof to apromoter, and incorporating the construct into an expression vector. Thevectors to be used are suitable for replication and, optionally,integration in eukaryotic cells. Typical vectors contain transcriptionand translation terminators, initiation sequences, and promoters usefulfor regulation of the expression of the desired nucleic acid sequence.

The vectors of the present invention may also be used for nucleic acidimmunization and gene therapy, using standard gene delivery protocols.Methods for gene delivery are known in the art. See, e.g., U.S. Pat.Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference hereinin their entireties. In another embodiment, the invention provides agene therapy vector.

The isolated nucleic acid of the invention can be cloned into a numberof types of vectors. For example, the nucleic acid can be cloned into avector including, but not limited to a plasmid, a phagemid, a phagederivative, an animal virus, and a cosmid. Vectors of particularinterest include expression vectors, replication vectors, probegeneration vectors, and sequencing vectors.

Further, the vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

For example, vectors derived from retroviruses such as the lentivirusare suitable tools to achieve long-term gene transfer since they allowlong-term, stable integration of a transgene and its propagation indaughter cells. Lentiviral vectors have the added advantage over vectorsderived from onco-retroviruses such as murine leukemia viruses in thatthey can transduce non-proliferating cells, such as hepatocytes. Theyalso have the added advantage of low immunogenicity. In a preferredembodiment, the composition includes a vector derived from anadeno-associated virus (AAV). Adeno-associated viral (AAV) vectors havebecome powerful gene delivery tools for the treatment of variousdisorders. AAV vectors possess a number of features that render themideally suited for gene therapy, including a lack of pathogenicity,minimal immunogenicity, and the ability to transduce postmitotic cellsin a stable and efficient manner. Expression of a particular genecontained within an AAV vector can be specifically targeted to one ormore types of cells by choosing the appropriate combination of AAVserotype, promoter, and delivery method

In one embodiment, the myotubularin encoding sequence is containedwithin an AAV vector. More than 30 naturally occurring serotypes of AAVare available. Many natural variants in the AAV capsid exist, allowingidentification and use of an AAV with properties specifically suited forskeletal muscle. AAV viruses may be engineered using conventionalmolecular biology techniques, making it possible to optimize theseparticles for cell specific delivery of myotubularin nucleic acidsequences, for minimizing immunogenicity, for tuning stability andparticle lifetime, for efficient degradation, for accurate delivery tothe nucleus, etc.

Thus, myotubularin overexpression can be achieved in the skeletal muscleby delivering a recombinantly engineered AAV or artificial AAV thatcontains sequences encoding myotubularin. The use of AAVs is a commonmode of exogenous delivery of DNA as it is relatively non-toxic,provides efficient gene transfer, and can be easily optimized forspecific purposes. Among the serotypes of AAVs isolated from human ornon-human primates (NHP) and well characterized, human serotype 2 is thefirst AAV that was developed as a gene transfer vector; it has beenwidely used for efficient gene transfer experiments in different targettissues and animal models. Clinical trials of the experimentalapplication of AAV2 based vectors to some human disease models are inprogress, and include therapies for diseases such as for example, cysticfibrosis and hemophilia B. Other useful AAV serotypes include AAV1,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9.

Desirable AAV fragments for assembly into vectors include the capproteins, including the vp1, vp2, vp3 and hypervariable regions, the repproteins, including rep 78, rep 68, rep 52, and rep 40, and thesequences encoding these proteins. These fragments may be readilyutilized in a variety of vector systems and host cells. Such fragmentsmay be used alone, in combination with other AAV serotype sequences orfragments, or in combination with elements from other AAV or non-AAVviral sequences. As used herein, artificial AAV serotypes include,without limitation, AAV with a non-naturally occurring capsid protein.Such an artificial capsid may be generated by any suitable technique,using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein)in combination with heterologous sequences which may be obtained from adifferent selected AAV serotype, non-contiguous portions of the same AAVserotype, from a non-AAV viral source, or from a non-viral source. Anartificial AAV serotype may be, without limitation, a chimeric AAVcapsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Thusexemplary AAVs, or artificial AAVs, suitable for expression ofmyotubularin, include AAV2/8 (see U.S. Pat. No. 7,282,199), AAV2/5(available from the National Institutes of Health), AAV2/9(International Patent Publication No. WO2005/033321), AAV2/6 (U.S. Pat.No. 6,156,303), and AAVrh8 (International Patent Publication No.WO2003/042397), among others.

In one embodiment, the vectors useful in the compositions and methodsdescribed herein contain, at a minimum, sequences encoding a selectedAAV serotype capsid, e.g., an AAV8 capsid, or a fragment thereof. Inanother embodiment, useful vectors contain, at a minimum, sequencesencoding a selected AAV serotype rep protein, e.g., AAV8 rep protein, ora fragment thereof. Optionally, such vectors may contain both AAV capand rep proteins. In vectors in which both AAV rep and cap are provided,the AAV rep and AAV cap sequences can both be of one serotype origin,e.g., all AAV8 origin. Alternatively, vectors may be used in which therep sequences are from an AAV serotype which differs from that which isproviding the cap sequences. In one embodiment, the rep and capsequences are expressed from separate sources (e.g., separate vectors,or a host cell and a vector). In another embodiment, these rep sequencesare fused in frame to cap sequences of a different AAV serotype to forma chimeric AAV vector, such as AAV2/8 described in U.S. Pat. No.7,282,199.

The AAV vectors of the invention further contain a minigene comprising amyotubularin nucleic acid sequence as described above which is flankedby AAV 5′ (inverted terminal repeat) ITR and AAV 3′ ITR.

A suitable recombinant adeno-associated virus (AAV) is generated byculturing a host cell which contains a nucleic acid sequence encoding anadeno-associated virus (AAV) serotype capsid protein, or fragmentthereof, as defined herein; a functional rep gene; a minigene composedof, at a minimum, AAV inverted terminal repeats (ITRs) and amyotubularin nucleic acid sequence, or biologically functional fragmentthereof; and sufficient helper functions to permit packaging of theminigene into the AAV capsid protein. The components required to becultured in the host cell to package an AAV minigene in an AAV capsidmay be provided to the host cell in trans. Alternatively, any one ormore of the required components (e.g., minigene, rep sequences, capsequences, and/or helper functions) may be provided by a stable hostcell which has been engineered to contain one or more of the requiredcomponents using methods known to those of skill in the art.

In specific embodiments, such a stable host cell will contain therequired component(s) under the control of a constitutive promoter. Inother embodiments, the required component(s) may be under the control ofan inducible promoter. Examples of suitable inducible and constitutivepromoters are provided elsewhere herein, and are well known in the art.In still another alternative, a selected stable host cell may containselected component(s) under the control of a constitutive promoter andother selected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contains the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transfersthe sequences carried thereon. The selected genetic element may bedelivered using any suitable method, including those described hereinand any others available in the art. The methods used to construct anyembodiment of this invention are known to those with skill in nucleicacid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques (see, e.g., Sambrook et al,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.). Similarly, methods of generating rAAV virions arewell known and the selection of a suitable method is not a limitation onthe present invention (see, e.g., K. Fisher et al, 1993 J. Virol.,70:520-532 and U.S. Pat. No. 5,478,745, among others).

Unless otherwise specified, the AAV ITRs, and other selected AAVcomponents described herein, may be readily selected from among any AAVserotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9 or other known or as yet unknown AAV serotypes.These ITRs or other AAV components may be readily isolated from an AAVserotype using techniques available to those of skill in the art. Suchan AAV may be isolated or obtained from academic, commercial, or publicsources (e.g., the American Type Culture Collection, Manassas, Va.).Alternatively, the AAV sequences may be obtained through synthetic orother suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank,PubMed, or the like.

The minigene is composed of, at a minimum, a myotubularin encodingnucleic acid sequence (the transgene) and its regulatory sequences, and5′ and 3′ AAV inverted terminal repeats (ITRs). In one desirableembodiment, the ITRs of AAV serotype 2 are used. However, ITRs fromother suitable serotypes may be selected. It is this minigene which ispackaged into a capsid protein and delivered to a selected host cell.The myotubularin encoding nucleic acid coding sequence is operativelylinked to regulatory components in a manner which permits transgenetranscription, translation, and/or expression in a host cell.

In addition to the major elements identified above for the minigene, theAAV vector also includes conventional control elements which areoperably linked to the transgene in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest. Expression control sequences includeappropriate transcription initiation, termination, promoter and enhancersequences; efficient RNA processing signals such as splicing andpolyadenylation (polyA) signals; sequences that stabilize cytoplasmicmRNA; sequences that enhance translation efficiency (i.e., Kozakconsensus sequence); sequences that enhance protein stability; and whendesired, sequences that enhance secretion of the encoded product. Agreat number of expression control sequences, including promoters whichare native, constitutive, inducible and/or tissue-specific, are known inthe art and may be utilized.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 by upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50by apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter. In one embodiment, the vector of the invention comprises atissue-specific promoter to drive expression of myotubularin in one ormore specific types of cells. In one embodiment, the vector of theinvention comprises a tissue-specific promoter to drive expression ofmyotubularin specifically in muscle. Tissue-specific promoters, whichcan be included in the vector of the invention to drive expressionspecifically in muscle are known in the art, and include, but are notlimited to a desmin promoter, myoglobin promoter, muscle creatine kinasepromoter, mammalian troponin 1 promoter, and skeletal alpha-actionpromoter. For example, in one embodiment, the vector comprises thedesmin promoter to provide expression of myotubularin specifically inmuscle cells.

Enhancer sequences found on a vector also regulates expression of thegene contained therein. Typically, enhancers are bound with proteinfactors to enhance the transcription of a gene. Enhancers may be locatedupstream or downstream of the gene it regulates. Enhancers may also betissue-specific to enhance transcription in a specific cell or tissuetype. In one embodiment, the vector of the present invention comprisesone or more enhancers to boost transcription of the gene present withinthe vector. For example, in one embodiment, the vector of the inventioncomprises a muscle-specific enhancer to enhance myotubularin expressionspecifically in muscle. Tissue-specific enhancers, which can be includedin the vector of the invention to drive expression specifically inmuscle are known in the art, and include, but are not limited to adesmin enhancer, muscle creatine kinase enhancer, myosin light-chainenhancer, myoglobin enhancer, and mammalian troponin 1 internalregulatory element.

In order to assess the expression of myotubularin, the expression vectorto be introduced into a cell can also contain either a selectable markergene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other aspects, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In one embodiment, the composition comprises a naked isolated nucleicacid encoding myotubularin, or a biologically functional fragmentthereof, wherein the isolated nucleic acid is essentially free fromtransfection-facilitating proteins, viral particles, liposomalformulations and the like (see, for example U.S. Pat. No. 5,580,859). Itis well known in the art that the use of naked isolated nucleic acidstructures, including for example naked DNA, works well with inducingexpression in muscle. As such, the present invention encompasses the useof such compositions for local delivery to the muscle and for systemicadministration (Wu et al., 2005, Gene Ther, 12(6): 477-486).

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

In one embodiment, the composition of the present invention comprises apeptide comprising myotubularin protein, or biologically functionalfragment thereof. The peptide of the present invention may be made usingchemical methods. For example, peptides can be synthesized by solidphase techniques (Roberge J Y et al (1995) Science 269: 202-204),cleaved from the resin, and purified by preparative high performanceliquid chromatography. Automated synthesis may be achieved, for example,using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordancewith the instructions provided by the manufacturer.

The invention should also be construed to include any form of a peptidehaving substantial homology to the peptides disclosed herein.Preferably, a peptide which is “substantially homologous” is about 50%homologous, more preferably about 70% homologous, even more preferablyabout 80% homologous, more preferably about 90% homologous, even morepreferably, about 95% homologous, and even more preferably about 99%homologous to amino acid sequence of the peptides disclosed herein.

The peptide may alternatively be made by recombinant means or bycleavage from a longer polypeptide. The composition of a peptide may beconfirmed by amino acid analysis or sequencing.

The variants of the polypeptides according to the present invention maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, (ii) onein which there are one or more modified amino acid residues, e.g.,residues that are modified by the attachment of substituent groups,(iii) one in which the polypeptide is an alternative splice variant ofthe polypeptide of the present invention, (iv) fragments of thepolypeptides and/or (v) one in which the polypeptide is fused withanother polypeptide, such as a leader or secretory sequence or asequence which is employed for purification (for example, His-tag) orfor detection (for example, Sv5 epitope tag). The fragments includepolypeptides generated via proteolytic cleavage (including multi-siteproteolysis) of an original sequence. Variants may bepost-translationally, or chemically modified. Such variants are deemedto be within the scope of those skilled in the art from the teachingherein.

As known in the art the “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to a sequence of a secondpolypeptide. Variants are defined to include polypeptide sequencesdifferent from the original sequence, preferably different from theoriginal sequence in less than 40% of residues per segment of interest,more preferably different from the original sequence in less than 25% ofresidues per segment of interest, more preferably different by less than10% of residues per segment of interest, most preferably different fromthe original protein sequence in just a few residues per segment ofinterest and at the same time sufficiently homologous to the originalsequence to preserve the functionality of the original sequence and/orthe ability to bind to ubiquitin or to a ubiquitylated protein. Thepresent invention includes amino acid sequences that are at least 60%,65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical tothe original amino acid sequence. The degree of identity between twopolypeptides is determined using computer algorithms and methods thatare widely known for the persons skilled in the art. The identitybetween two amino acid sequences is preferably determined by using theBLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990)].

The polypeptides of the invention can be post-translationally modified.For example, post-translational modifications that fall within the scopeof the present invention include signal peptide cleavage, glycosylation,acetylation, isoprenylation, proteolysis, myristoylation, proteinfolding and proteolytic processing, etc. Some modifications orprocessing events require introduction of additional biologicalmachinery. For example, processing events, such as signal peptidecleavage and core glycosylation, are examined by adding caninemicrosomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489)to a standard translation reaction.

The polypeptides of the invention may include unnatural amino acidsformed by post-translational modification or by introducing unnaturalamino acids during translation. A variety of approaches are availablefor introducing unnatural amino acids during protein translation.

The term “functionally equivalent” as used herein refers to apolypeptide according to the invention that preferably retains at leastone biological function or activity of the specific amino acid sequenceof myotubularin.

A peptide or protein of the invention may be conjugated with othermolecules, such as proteins, to prepare fusion proteins. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins provided that the resulting fusion protein retains thefunctionality of the myotubularin comprising peptide.

A peptide or protein of the invention may be phosphorylated usingconventional methods such as the method described in Reedijk et al. (TheEMBO Journal 11(4):1365, 1992).

Cyclic derivatives of the peptides or chimeric proteins of the inventionare also part of the present invention. Cyclization may allow thepeptide or chimeric protein to assume a more favorable conformation forassociation with other molecules. Cyclization may be achieved usingtechniques known in the art. For example, disulfide bonds may be formedbetween two appropriately spaced components having free sulfhydrylgroups, or an amide bond may be formed between an amino group of onecomponent and a carboxyl group of another component. Cyclization mayalso be achieved using an azobenzene-containing amino acid as describedby Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. Thecomponents that form the bonds may be side chains of amino acids,non-amino acid components or a combination of the two. In an embodimentof the invention, cyclic peptides may comprise a beta-turn in the rightposition. Beta-turns may be introduced into the peptides of theinvention by adding the amino acids Pro-Gly at the right position.

It may be desirable to produce a cyclic peptide which is more flexiblethan the cyclic peptides containing peptide bond linkages as describedabove. A more flexible peptide may be prepared by introducing cysteinesat the right and left position of the peptide and forming a disulphidebridge between the two cysteines. The two cysteines are arranged so asnot to deform the beta-sheet and turn. The peptide is more flexible as aresult of the length of the disulfide linkage and the smaller number ofhydrogen bonds in the beta-sheet portion. The relative flexibility of acyclic peptide can be determined by molecular dynamics simulations.

(a) Tags: In a particular embodiment of the invention, the polypeptideof the invention further comprises the amino acid sequence of a tag. Thetag includes but is not limited to: polyhistidine tags (His-tags) (forexample H6 and H10, etc.) or other tags for use in IMAC systems, forexample, Ni²⁺ affinity columns, etc., GST fusions, MBP fusions,streptavidine-tags, the BSP biotinylation target sequence of thebacterial enzyme BIRA and tag epitopes that are directed by antibodies(for example c-myc tags, FLAG-tags, among others). As will be observedby a person skilled in the art, the tag peptide can be used forpurification, inspection, selection and/or visualization of the fusionprotein of the invention. In a particular embodiment of the invention,the tag is a detection tag and/or a purification tag. It will beappreciated that the tag sequence will not interfere in the function ofthe protein of the invention.

(b) Leader and secretory sequences: Accordingly, the polypeptides of theinvention can be fused to another polypeptide or tag, such as a leaderor secretory sequence or a sequence which is employed for purificationor for detection. In a particular embodiment, the polypeptide of theinvention comprises the glutathione-S-transferase protein tag whichprovides the basis for rapid high-affinity purification of thepolypeptide of the invention. Indeed, this GST-fusion protein can thenbe purified from cells via its high affinity for glutathione. Agarosebeads can be coupled to glutathione, and such glutathione-agarose beadsbind GST-proteins. Thus, in a particular embodiment of the invention,the polypeptide of the invention is bound to a solid support. In apreferred embodiment, if the polypeptide of the invention comprises aGST moiety, the polypeptide is coupled to a glutathione-modifiedsupport. In a particular case, the glutathione modified support is aglutathione-agarose bead. Additionally, a sequence encoding a proteasecleavage site can be included between the affinity tag and thepolypeptide sequence, thus permitting the removal of the binding tagafter incubation with this specific enzyme and thus facilitating thepurification of the corresponding protein of interest.

(c) Targeting sequences: The invention also relates to peptidescomprising myotubularin fused to, or integrated into, a target protein,and/or a targeting domain capable of directing the chimeric protein to adesired cellular component or cell type or tissue. The chimeric proteinsmay also contain additional amino acid sequences or domains. Thechimeric proteins are recombinant in the sense that the variouscomponents are from different sources, and as such are not foundtogether in nature (i.e. are heterologous).

A target protein is a protein that is selected for degradation and forexample may be a protein that is mutated or over expressed in a diseaseor condition. In another embodiment of the invention, a target proteinis a protein that is abnormally degraded and for example may be aprotein that is mutated or underexpressed in a disease or condition. Thetargeting domain can be a membrane spanning domain, a membrane bindingdomain, or a sequence directing the protein to associate with forexample vesicles or with the nucleus. The targeting domain can target apeptide to a particular cell type or tissue. For example, the targetingdomain can be a cell surface ligand or an antibody against cell surfaceantigens of a target tissue (e.g. muscle tissue). A targeting domain maytarget the peptide of the invention to a cellular component.

(d) Intracellular targeting: Combined with certain formulations, suchpeptides can be effective intracellular agents. However, in order toincrease the efficacy of such peptides, the peptide of the invention canbe provided a fusion peptide along with a second peptide which promotes“transcytosis”, e.g., uptake of the peptide by epithelial cells. Toillustrate, the peptide of the present invention can be provided as partof a fusion polypeptide with all or a fragment of the N-terminal domainof the HIV protein Tat, e.g., residues 1-72 of Tat or a smaller fragmentthereof which can promote transcytosis. In other embodiments, the RLPcan be provided a fusion polypeptide with all or a portion of theantenopedia III protein.

To further illustrate, the peptide of the invention can be provided as achimeric peptide which includes a heterologous peptide sequence(“internalizing peptide”) which drives the translocation of anextracellular form of the peptide across a cell membrane in order tofacilitate intracellular localization of the peptide. In this regard,the peptide is one which is active intracellularly. The internalizingpeptide, by itself, is capable of crossing a cellular membrane by, e.g.,transcytosis, at a relatively high rate. The internalizing peptide isconjugated, e.g., as a fusion protein, to a peptide comprisingmyotubularin. The resulting chimeric peptide is transported into cellsat a higher rate relative to the peptide alone to thereby provide ameans for enhancing its introduction into cells to which it is applied.

(e) Peptide Mimetics:

In other embodiments, the subject compositions are peptidomimetics ofthe peptide of the invention. Peptidomimetics are compounds based on, orderived from, peptides and proteins. The peptidomimetics of the presentinvention typically can be obtained by structural modification of aknown sequence using unnatural amino acids, conformational restraints,isosteric replacement, and the like. The subject peptidomimeticsconstitute the continum of structural space between peptides andnon-peptide synthetic structures; peptidomimetics may be useful,therefore, in delineating pharmacophores and in helping to translatepeptides into nonpeptide compounds with the activity of the parentpeptides.

Moreover, as is apparent from the present disclosure, mimetopes of thesubject peptides can be provided. Such peptidomimetics can have suchattributes as being non-hydrolyzable (e.g., increased stability againstproteases or other physiological conditions which degrade thecorresponding peptide), increased specificity and/or potency, andincreased cell permeability for intracellular localization of thepeptidomimetic. For illustrative purposes, peptide analogs of thepresent invention can be generated using, for example, benzodiazepines(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p 123), C-7mimics (Huffman et al. in Peptides: Chemistry and Biologyy, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231),β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71),diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun124:141), and methyleneamino-modifed (Roark et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988, p 134). Also, see generally, Session III: Analyticand synthetic methods, in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988)

In addition to a variety of side chain replacements which can be carriedout to generate the peptidomimetics, the present invention specificallycontemplates the use of conformationally restrained mimics of peptidesecondary structure. Numerous surrogates have been developed for theamide bond of peptides. Frequently exploited surrogates for the amidebond include the following groups (i) trans-olefins, (ii) fluoroalkene,(iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.

Moreover, other examples of mimetopes include, but are not limited to,protein-based compounds, carbohydrate-based compounds, lipid-basedcompounds, nucleic acid-based compounds, natural organic compounds,synthetically derived organic compounds, anti-idiotypic antibodiesand/or catalytic antibodies, or fragments thereof. A mimetope can beobtained by, for example, screening libraries of natural and syntheticcompounds for compounds capable of binding to the peptide of theinvention. A mimetope can also be obtained, for example, from librariesof natural and synthetic compounds, in particular, chemical orcombinatorial libraries (i.e., libraries of compounds that differ insequence or size but that have the same building blocks). A mimetope canalso be obtained by, for example, rational drug design. In a rationaldrug design procedure, the three-dimensional structure of a compound ofthe present invention can be analyzed by, for example, nuclear magneticresonance (NMR) or x-ray crystallography. The three-dimensionalstructure can then be used to predict structures of potential mimetopesby, for example, computer modeling, the predicted mimetope structurescan then be produced by, for example, chemical synthesis, recombinantDNA technology, or by isolating a mimetope from a natural source (e.g.,plants, animals, bacteria and fungi).

A peptide of the invention may be synthesized by conventionaltechniques. For example, the peptides or chimeric proteins may besynthesized by chemical synthesis using solid phase peptide synthesis.These methods employ either solid or solution phase synthesis methods(see for example, J. M. Stewart, and J. D. Young, Solid Phase PeptideSynthesis, 2^(nd) Ed., Pierce Chemical Co., Rockford Ill. (1984) and G.Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biologyeditors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York,1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, suprs, Vol 1, for classical solution synthesis.) By way ofexample, a RLP or chimeric protein may be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry with direct incorporationof phosphothreonine as theN-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

N-terminal or C-terminal fusion proteins comprising a peptide orchimeric protein of the invention conjugated with other molecules may beprepared by fusing, through recombinant techniques, the N-terminal orC-terminal of the peptide or chimeric protein, and the sequence of aselected protein or selectable marker with a desired biologicalfunction. The resultant fusion proteins contain the myotubularincomprising peptide or chimeric protein fused to the selected protein ormarker protein as described herein. Examples of proteins which may beused to prepare fusion proteins include immunoglobulins,glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

Peptides of the invention may be developed using a biological expressionsystem. The use of these systems allows the production of largelibraries of random peptide sequences and the screening of theselibraries for peptide sequences that bind to particular proteins.Libraries may be produced by cloning synthetic DNA that encodes randompeptide sequences into appropriate expression vectors. (see Christian etal 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404;Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries mayalso be constructed by concurrent synthesis of overlapping peptides (seeU.S. Pat. No. 4,708,871).

The peptides and chimeric proteins of the invention may be convertedinto pharmaceutical salts by reacting with inorganic acids such ashydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid,etc., or organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid,malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid,benezenesulfonic acid, and toluenesulfonic acids.

Dosage and Formulation (Pharmaceutical Compositions)

The present invention envisions treating a disease, for example,myopathy and the like, in a subject by the administration of therapeuticagent, e.g. a composition comprising MTM1.

Administration of the therapeutic agent or modified cell in accordancewith the present invention may be continuous or intermittent, depending,for example, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of the agentsor modified cell of the invention may be essentially continuous over apreselected period of time or may be in a series of spaced doses. Bothlocal and systemic administration is contemplated. The amountadministered will vary depending on various factors including, but notlimited to, the composition chosen, the particular disease, the weight,the physical condition, and the age of the mammal, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems which are well known to the art

One or more suitable unit dosage forms having the therapeutic agent(s)of the invention, which, as discussed below, may optionally beformulated for sustained release (for example using microencapsulation,see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of whichare incorporated by reference herein), can be administered by a varietyof routes including parenteral, including by intravenous andintramuscular routes, as well as by direct injection into the diseasedtissue. For example, the therapeutic agent or modified cell may bedirectly injected into the muscle. The formulations may, whereappropriate, be conveniently presented in discrete unit dosage forms andmay be prepared by any of the methods well known to pharmacy. Suchmethods may include the step of bringing into association thetherapeutic agent with liquid carriers, solid matrices, semi-solidcarriers, finely divided solid carriers or combinations thereof, andthen, if necessary, introducing or shaping the product into the desireddelivery system.

When the therapeutic agents of the invention are prepared foradministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules; as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual aerosol dose of each dosage formneed not in itself constitute an effective amount for treating theparticular indication or disease since the necessary effective amountcan be reached by administration of a plurality of dosage units.Moreover, the effective amount may be achieved using less than the dosein the dosage form, either individually, or in a series ofadministrations.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions, such as phosphate buffered saline solutionspH 7.0-8.0.

The expression vectors, transduced cells, polynucleotides andpolypeptides (active ingredients) of this invention can be formulatedand administered to treat a variety of disease states by any means thatproduces contact of the active ingredient with the agent's site ofaction in the body of the organism. They can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic active ingredients orin a combination of therapeutic active ingredients. They can beadministered alone, but are generally administered with a pharmaceuticalcarrier selected on the basis of the chosen route of administration andstandard pharmaceutical practice.

In general, water, suitable oil, saline, aqueous dextrose (glucose), andrelated sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration contain the active ingredient,suitable stabilizing agents and, if necessary, buffer substances.Antioxidizing agents such as sodium bisulfate, sodium sulfite orascorbic acid, either alone or combined, are suitable stabilizingagents. Also used are citric acid and its salts and sodiumEthylenediaminetetraacetic acid (EDTA). In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, astandard reference text in this field.

The active ingredients of the invention may be formulated to besuspended in a pharmaceutically acceptable composition suitable for usein mammals and in particular, in humans. Such formulations include theuse of adjuvants such as muramyl dipeptide derivatives (MDP) or analogsthat are described in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536;4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful,include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate anddimethyl-dioctadecylammonium bromide (DDA), Freund's adjuvant, andIL-12. Other components may include a polyoxypropylene-polyoxyethyleneblock polymer (Pluronic®), a non-ionic surfactant, and a metabolizableoil such as squalene (U.S. Pat. No. 4,606,918).

Additionally, standard pharmaceutical methods can be employed to controlthe duration of action. These are well known in the art and includecontrol release preparations and can include appropriate macromolecules,for example polymers, polyesters, polyamino acids, polyvinyl,pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethylcellulose or protamine sulfate. The concentration of macromolecules aswell as the methods of incorporation can be adjusted in order to controlrelease. Additionally, the agent can be incorporated into particles ofpolymeric materials such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. In addition to beingincorporated, these agents can also be used to trap the compound inmicrocapsules.

Accordingly, the pharmaceutical composition of the present invention maybe delivered via various routes and to various sites in an mammal bodyto achieve a particular effect (see, e.g., Rosenfeld et al., 1991;Rosenfeld et al., 1991 a; Jaffe et al., supra; Berkner, supra). Oneskilled in the art will recognize that although more than one route canbe used for administration, a particular route can provide a moreimmediate and more effective reaction than another route. Local orsystemic delivery can be accomplished by administration comprisingapplication or instillation of the formulation into body cavities,inhalation or insufflation of an aerosol, or by parenteral introduction,comprising intramuscular, intravenous, peritoneal, subcutaneous,intradermal, as well as topical administration.

The active ingredients of the present invention can be provided in unitdosage form wherein each dosage unit, e.g., a teaspoonful, tablet,solution, or suppository, contains a predetermined amount of thecomposition, alone or in appropriate combination with other activeagents. The term “unit dosage form” as used herein refers to physicallydiscrete units suitable as unitary dosages for human and mammalsubjects, each unit containing a predetermined quantity of thecompositions of the present invention, alone or in combination withother active agents, calculated in an amount sufficient to produce thedesired effect, in association with a pharmaceutically acceptablediluent, carrier, or vehicle, where appropriate. The specifications forthe unit dosage forms of the present invention depend on the particulareffect to be achieved and the particular pharmacodynamics associatedwith the pharmaceutical composition in the particular host.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Gene Therapy Administration

One skilled in the art recognizes that different methods of delivery maybe utilized to administer a vector into a cell. Examples include: (1)methods utilizing physical means, such as electroporation (electricity),a gene gun (physical force) or applying large volumes of a liquid(pressure); and (2) methods wherein the vector is complexed to anotherentity, such as a liposome, aggregated protein or transporter molecule.

Furthermore, the actual dose and schedule can vary depending on whetherthe compositions are administered in combination with otherpharmaceutical compositions, or depending on interindividual differencesin pharmacokinetics, drug disposition, and metabolism. Similarly,amounts can vary in in vitro applications depending on the particularcell line utilized (e.g., based on the number of vector receptorspresent on the cell surface, or the ability of the particular vectoremployed for gene transfer to replicate in that cell line). Furthermore,the amount of vector to be added per cell will likely vary with thelength and stability of the therapeutic gene inserted in the vector, aswell as also the nature of the sequence, and is particularly a parameterwhich needs to be determined empirically, and can be altered due tofactors not inherent to the methods of the present invention (forinstance, the cost associated with synthesis). One skilled in the artcan easily make any necessary adjustments in accordance with theexigencies of the particular situation.

Cells containing the therapeutic agent may also contain a suicide genei.e., a gene which encodes a product that can be used to destroy thecell. In many gene therapy situations, it is desirable to be able toexpress a gene for therapeutic purposes in a host, cell but also to havethe capacity to destroy the host cell at will. The therapeutic agent canbe linked to a suicide gene, whose expression is not activated in theabsence of an activator compound. When death of the cell in which boththe agent and the suicide gene have been introduced is desired, theactivator compound is administered to the cell thereby activatingexpression of the suicide gene and killing the cell. Examples of suicidegene/prodrug combinations which may be used are herpes simplexvirus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir;oxidoreductase and cycloheximide; cytosine deaminase and5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) andAZT; and deoxycytidine kinase and cytosine arabinoside.

Therapeutic

The present invention encompasses a method to treat myopathy in asubject diagnosed with a myopathy or in a subject at risk for developinga myopathy. The method improves muscle strength and muscle function inthose in need thereof. Further, the method improves quality of life andprolongs survival in a patient with a myopathy. In one embodiment, themethod of the present invention comprises administering to a subject acomposition comprising the MTM1 gene or functional fragment thereof. Inanother embodiment, the method of the present invention comprisesadministering to a subject a composition comprising a nucleic acidsequence encoding myotubularin. In another embodiment, the methodcomprises inducing the expression of myotubularin specifically in themuscle of the subject. In one embodiment, the subject is a mammal.Preferably the mammal is a human.

The method of the present invention is used to treat any type ofmyopathy in a subject. A myopathy is a muscular disease wherein themuscle fibers of an afflicted subject are not functioning normally.Non-functional, partially-functional, or sub-optimally-functional musclefibers lead to overall muscle weakness, which may result in loss ofmotor function and respiratory control. In one embodiment, the method ofthe present invention is used to treat acute myopathies that may occur,for example, as a symptom in systemic disease processes. In anotherembodiment, the method of the present invention is used to treat chronicmyopathy, including inherited myopathies or dystrophies.

Muscular dystrophies are a subgroup of myopathy characterized,generally, by progressive weakening of muscle tissue through increasedmuscle degeneration and reduced muscle regeneration. Musculardystrophies are generally inherited, with specific forms of musculardystrophy following specific patterns of inheritance. Forms of musculardystrophy include Duchenne muscular dystrophy, Becker's musculardystrophy, congenital muscular dystrophy, distal muscular dystrophy,distal muscular dystrophy, Miyoshi myopathy, Limb Girdle MuscularDystrophy, Emery-Dreifuss muscular dystrophy, Facioscapulohumeralmuscular dystrophy, myotonic muscular dystrophy, and oculopharyngealmuscular dystrophy.

Congential myopathies are another subgroup of inherited myopathies thatdo not include progressive muscle death, as seen in dystrophies, butrather is associated with reduced contraction of the muscle tissue.Forms of congenital myopathy include namaline myopathy, multi/minicoremyopathy, and myotubular myopathy. Myotubular myopathy is a raredisorder that in many cases presents in infants, with afflicted subjectshaving low muscle tone, severe weakness, delayed development, andpulmonary complications. Inheritance of myotubular myopathy includesforms that are X-linked, autosomal recessive, and autosomal dominant.The X-linked form (XLMTM) is the most common form of myotubularmyopathy, and has a life expectancy of only a few years. XLMTM is causedby genetic mutations in the MTM1 gene, with the severity of the diseasedependent on the particular type of mutation. The present invention isbased upon the delivery of functional MTM1, which thereby restoresproper muscle function. As described elsewhere herein, administration ofMTM1 improves muscle function and prolongs survival in afflictedsubjects.

The present method is not limited to treatment of any particularmyopathy, as it is contemplated herein that enhanced expression ofmyotubularin improves muscle function of a wide variety of disorders.Further, the present method is not limited to treatment of a subject whois clinically diagnosed with any particular disorder. For example, inone embodiment, the method comprises administering a compositioncomprising MTM1 to a subject in need of improved muscle function. Thiscan include subjects who have been bedridden or otherwise immobile,causing their muscles to atrophy.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials. When “an effective amount” or “therapeutic amount” isindicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, diseaseprogression, and condition of the patient (subject). The optimal dosageand treatment regime for a particular patient can readily be determinedby one skilled in the art of medicine by monitoring the patient forsigns of disease and adjusting the treatment accordingly.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the method of the inventioncomprises a systemic administration of a composition comprising MTM1. Incertain embodiments, systemic delivery comprises administration at ornear weakened or afflicted muscle. For example, in one embodiment,systemic delivery comprises intravenous delivery near a weakened tissue.However, it is demonstrated herein that this delivery produces regionalincreases in muscle function, at or near the site of delivery, as wellas global increases in muscle functions in other parts of the body.Thus, in certain embodiments, the invention comprises a local deliveryof the composition which produces systemic effects. In otherembodiments, systemic delivery comprises administration at a distancefrom any weakened or afflicted muscle. In certain instances, a systemicdelivery is preferred as it is demonstrated herein that systemicdelivery of MTM1 effectively improves regional and global musclefunction throughout the subject. Thus, systemic delivery eliminates theneed for a multitude of local deliveries, and allows for efficientinduction of myotubularin expression in muscle tissue that may bedifficult to reach using local delivery methods (e.g. diaphragm). Asdemonstrated herein, systemic delivery improves muscle function in theaffected diaphragm. Thus, systemic delivery of the composition describedherein improves respiratory function without the need for directinjection into the diaphragm. This thereby provides an easy,non-invasive and effective treatment method that can vastly improvesurvival and quality of life of an afflicted subject.

In certain embodiments of the present invention, the composition, asdescribed herein, are administered to a subject in conjunction with(e.g. before, simultaneously, or following) any number of relevanttreatment modalities, including but not limited to immunosuppressiveagents, supportive therapy, respiratory support, nutritional support,orthopedic support, and the like.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Gene Replacement Therapy Prolongs Survival and Restores MuscleFunction in Murine and Canine Models of X-Linked Myotubular Myopathy

Loss-of-function mutations in the myotubularin gene (MTM1) causeX-linked myotubular myopathy (XLMTM), a fatal pediatric disease ofskeletal muscle with no effective treatment. Described herein isexamination of gene therapy in mouse and dog models of XLMTM. Systemicdelivery of a single dose of a recombinant adeno-associated virus (rAAV)vector expressing murine myotubularin in Mtm1-deficient knockout miceresulted in robust improvement in motor activity and contractile force,attenuated pathology and prolonged survival from under two months to atleast one year. Intramuscular and intravascular rAAV-mediated deliveryof canine MTM1 in affected XLMTM dogs resulted in similar robustimprovement in muscle contractile force, attenuated pathology andprolonged survival. The results presented herein demonstrate thatmyotubularin gene delivery rescues severe muscle pathology in mutantanimals, providing proof-of-concept for future clinical trials in XLMTMpatients.

It was examined whether restoration of functional myotubularin wouldameliorate the severe muscular weakness. Described herein is thelong-term impact of systemic delivery of a rAAV-Mtm1 vector inmyotubularin-deficient mice.

To bridge the translational gap between mice and men, a canine breedingcolony was established from a female Labrador Retriever carrying anX-linked MTM1 missense mutation (Beggs et al., 2010, Proc Natl Acad SciUSA 107(33):14697-702). Muscles from affected males exhibit profoundquantitative reduction and altered localization of myotubularin, likelydue to sequestration and degradation of misfolded protein. Thephysiological defects closely resemble those of humans with comparablysevere mutations. Reported herein are the results of rAAV-mediatedmyotubularin transduction in limb muscles of six XLMTM dogs as the firstlarge animal predictive model of gene therapy for this disease.

The materials and methods employed in these experiments are nowdescribed.

Animals

The constitutive (whole-body) knockout of the myotubularin gene(KO-Mtm1, also named BS53d4-129pas) and the muscle-specific knockout(mKO-Mtm1) on the C57BL/6 background were described previously(Buj-Bello et al., 2002, Proc Natl Acad Sci USA 99(23):15060-5;Al-Qusairi et al., 2009, Proc Natl Acad Sci USA 106(44):18763-8).Wild-type littermate males were used as controls.

XLMTM dogs were described previously (Beggs et al., 2010, Proc Natl AcadSci USA 107(33):14697-702). Affected males were identified by polymerasechain reaction-based genotyping, as described.

Preparation and Intravascular Delivery rAAV-Mtm1 in Mice

The recombinant adeno-associated virus vectorsrAAV2/8-pDesmin-Mtm1^(murine) and rAAV2/9-pDesmin-Mtm1^(murine) wereconstructed as follows. Murine Mtm1 cDNA (AF073996, NCBI) was cloneddownstream of the human desmin promoter (Wang et al., Mol Ther15(6):1160-6) in the AAV2 expression plasmid pAAV2-pDes by PCRamplification. All clones were verified by DNA sequencing. Pseudo-typedrecombinant rAAV2/8 and rAAV2/9 viral preparations were generated bypackaging AAV2-inverted terminal repeat (ITR) recombinant genomes intoAAV8 or AAV9 capsids. Briefly, the cis-acting plasmid encoding thetransgene pAAV-pDesmin-Mtm1, the trans-complementing rep-cap9 plasmidencoding the proteins necessary for the replication and the structure ofvector and the adenovirus helper plasmid cells were transfected togetherinto HEK293 cells. After three days, both the culture supernatant andthe monolayer cells were harvested and cells were broken by repetitivefreeze-and-thaw cycles. Vector particles were purified through twosequential rounds of CsCl gradient ultra-centrifugation and dialyzedagainst sterile PBS. Viral titers were quantified by a TAQMAN® real-timePCR assay (Applied Biosystem) with primers and probes specific for theITR2 regions (Qiao et al., 2009, Hum Gene Ther 20(1):1-10) and expressedas viral genomes per ml (vg/ml).

rAAV-Mtm1 at 3×10¹³ viral genomes per kg body mass [(vg/kg)] wasinjected into the tail vein of 3- and 5-week-old KO-Mtm1 mice. Anequivalent volume of saline was administrated to either KO-Mtm1 orwild-type (WT) animals as controls. The same vector (0.5×10¹³ vg/kg) oran equivalent volume of saline was injected into the tail vein of 4week-old mKO-Mtm1 or WT littermate mice.

Preparation and Administration of rAAV8-MTM1 in Dogs

The recombinant adeno-associated virus vector containing a caninemyotubularin cDNA regulated by the desmin promoter,rAAV2/8-pDesmin-MTM1^(canine) (designated rAAV8-MTM1), was produced in abaculovirus/Sf9 system. Two baculovirus batches were generated, oneexpressing rep and cap AAV genes and the second bearing the canine MTM1cDNA (XM850116, NCBI) downstream from the human desmin promoter(pDesmin). The rAAV-MTM1 vector particles were produced afterbaculoviral double infection of insect Sf9 cells and purified from totalcell culture using AVB affinity chromatography column (GE Healthcare,AVB SEPHAROSE® high performance). The concentration in vg/mL wasdetermined from DNase-resistant particles, as described above. Otherroutine quality control assays for rAAV vectors were performed,including sterility and purity tests (Yuasa et al., 2007, Gene Ther14(17):1249-60).

Intramuscular Injections.

rAAV8-MTM1 (4×10¹¹ vg) diluted in 1 ml lactated Ringer's solution wasinjected under ultrasound guidance into the midbelly of the cranialtibialis muscle of one hind limb of unvaccinated 10 weeks-of-ageaffected male XLMTM dogs under anesthesia. The cranial tibialis muscleof the contralateral limb was injected with an equal volume of Ringer'ssolution alone. Unaffected male littermates (WT) received 1 ml ofRinger's solution in each hind limb.

Intravenous Regional Limb Infusions.

In anesthetized XLMTM dogs rAAV8-MTM1 (2.5×10¹³ vg/kg) diluted inphosphate buffered saline (PBS) was infused into the distal saphenousvein under pressure (300 torr) against a tourniquet as described (Petrovet al., 2011, Methods Mol Biol 709:277-86; Arruda et al., 2010, Blood115(23):4678-88). Briefly, a tourniquet was positioned at the level ofthe groin and adjusted until the femoral pulse was no longer detectableby ultrasound to transiently block blood inflow to the target limb. Atight extensible wrap applied in a distal to proximal directionexsanguinated the limb before the tourniquet was tightened. Vector wassuspended in PBS at 20% of the total hind limb volume (determined bywater volume displacement) and administered via a 14 gauge catheterplaced into a distal branch of the peripheral saphenous vein on thedorsum of the paw. The tourniquet was tightened for a total of 15minutes (10 minutes prior to and 5 minutes during the infusion). In eachdog, one hind limb was infused with vector whereas the contralateralhind limb was not infused.

Mtm1 Nucleic Acid Sequences

Mtm1 nucleic acid sequences are as follows: mouse Mtm: SEQ ID NOs 1-2;canine MTM1: SEQ ID NOs 3-4; and human MTM1: SEQ ID NOs 5-6.

Vector Copy Number (VCN) Analysis

The number of vector genomes per diploid genome was quantified from 80ng of total DNA by TAQMAN® real-time PCR with a 7900 HT thermocycler(Applied Biosystems). The canine β glucuronidase gene was used forstandardization. The primers and probe used for vector genome (MTM1)amplification were as follows:

(forward, SEQ ID NO: 7) 5′-ATAAGTTTTGGACATAAGTTTGC-3′,(reverse, SEQ ID NO: 8) 5′-CATTTGCCATACACAATCAA-3′, and(probe, SEQ ID NO: 9) 5′-CGACGCTGACCGGTCTCCTA-3′.The primers (Applied Biosystems) and probe used for 0 glucuronidaseamplification were as follows:

(forward, SEQ ID NO: 10) 5′-ACGCTGATTGCTCACACCAA-3′,(reverse, SEQ ID NO: 11) 5′-CCCCAGGTCTGCTTCATAGTTG-3′, and(probe, SEQ ID NO: 12) 5′-CCCGGCCCGTGACCTTTGTGA-3′.Quantitative Immunoblot Analysis

Several muscle cryo-sections of thirty μm each (300 μm to 1 mm in total)were sliced and proteins were extracted using a lysis buffer containing10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Naorthovanadate, 100 mM NaF, 4 mM sodium pyrophosphate, 1% Triton X-100,0.5% IGEPAL, and Protease Inhibitor Cocktail used according to themanufacturer's instruction (Roche Applied Science). Samples wereincubated on ice with occasional mixing during 30 min. Aftercentrifugation at 10,000 g for 10 min at 4° C., the supernatants wererecovered for Western Blot analysis. Total protein concentration wasdetermined by the Bradford methodology according to the manufacturer'sinstruction (BioRad). Thirty to sixty μg of total proteins weredenatured for 5 min at 95° C. in a buffer containing 125 mM Tris.HCl pH6.8, 4% SDS, 0.2M DTT, 50% glycerol and bromophenol blue. Proteinsamples were submitted to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis in 10% acrylamide gels and transferred onto 0.2 μmnitrocellulose membranes (GE Healthcare) by the application of anelectric field (100 V, 1 hour) at 4° C. The membranes were firstincubated for 60 min at room temperature in a blocking solution composedof Tris-buffered-saline (TBS), 0.1% Tween 20 and 5% milk. For mousestudies, membranes were probed successively with a rabbit polyclonalantibody raised against the C-terminal extremity of murine myotubularin(R2348 (Buj-Bello et al., 2008, Hum Mol Genet 17(14):2132-43)) and amouse monoclonal antibody specific for GAPDH (Millipore, MAB374). Fordog studies, membranes were probed with a rabbit polyclonal antibodyraised against the C-terminus of canine myotubularin (R1040, Généthon).Antibody incubations were carried out overnight at 4° C. in TBS, 0.1%Tween 20 and 5% milk. Detection was performed with a secondary antibodycoupled to IRDye 680 (LI-COR) and the membranes were exposed to theODYSSEY® infrared imaging system (LI-COR Biotechnology Inc.) fordetection and quantification of the fluorescence signal (ODYSSEY®software). All gel electrophoresis, transfer and blotting procedureswere repeated to produce three independent immunoblots for each sample.ImageJ densitometry software (Version 1.4, National Institute of Health,Bethesda, Md.) was also used to measure density of immunoblot bands(Maulik and Thirunavukkarasu, 2008, J Mol Cell Cardioln 44(2):219-27;Tsunoda et al., 2008, Am J Physiol Endocrinol Metab 294(5):E833-40;Fukushima et al., 2007, BMC Musculoskelet Disord 8:54). Optical densityfor each band was measured three times and values were averaged.

Histology, Morphometry, Immunofluorescence, Electron Microscopy

For mouse studies, serial 7 μm thick transverse cryo-sections wereprepared from frozen muscles and processed for hematoxylin and eosin(HE) staining using standard procedures. The proportion of internalizednuclei was quantified from HE-stained sections using the Histolabsoftware (Microvision). For NADH-TR coloration muscle cryo-sections wereincubated for 10 min at 57° C. in a solution composed of 50 mM Tris HCl,pH 7.3, 1.2 mM Nitroblue Tetrazolium (sigma) and 0.6 mM β-NADH (Sigma),washed, and mounted in EUKITT® (Fluka). For determination of the numberand minimal diameter of myofibers, laminin immuno-staining was performedto delineate each fiber. Briefly, endogenous peroxidases wereneutralized by incubation in H₂O₂ for 30 min. Sections were incubatedfor 30 minutes with PBS/10% goat serum in order to block unspecificsites and then overnight in a 1:1,000 dilution of anti-laminin rabbitpolyclonal antibody (DAKO, Z0097) at room temperature. Sections werethen processed according to manufacturer's guidelines (Kit En Vision™Rabbit HRP, DAKO). Myofibers minor diameters were automatically measuredon digital images of the sections by the Ellix software (Microvision).

For immunofluorescence staining of mouse and dog muscle tissue,transverse sections were fixed for 10 min by incubation in PBS at 100°C. Non-specific antigens were blocked with PBS, 0.2% Tween, 3% BSA atRT. Sections were then incubated at 4° C. overnight with mouse primaryantibodies directed against DHPRla (Thermo Scientific Pierce, MA3-920)or Dysferlin (Novocastra, NCL-HAMLET). After extensive PBS washes,sections were incubated with biotinylated goat anti mouse antibodies(SouthernBiotech) and, after additional washes, with streptavidinconjugated with ALEXA FLUOR® 488 (Invitrogen). Glass slides were mountedwith FLUORSAVE™ reagent (Calbiochem®, Merck) and visualized with a Leicaconfocal microscope TCS-SP2. Digital images of a slice corresponding tothe muscle midsection were acquired with a CCD camera (Sony) and amotorized stage.

For histological studies of canine tissue following intramuscularinjection, serial 8 μm thick transverse cryo-sections were prepared fromfrozen mid tibialis anterior muscles and processed for hematoxylin andeosin (HE) and NADH-TR staining, as described above. The number ofcentrally nucleated fibers, and fibers with mislocalization oforganelles (including mitochondrial aggregates and necklace fibers) werequantified manually from photographs taken at 200× magnification. Thenumber of fibers assessed ranged from 200-861. For fiber sizequantification, muscles were immunostained as described above withrabbit anti-dystrophin antibodies (Abcam PLC, ab15277) and ALEXAFLUOR®-conjugated anti-rabbit IgG (Molecular Probes). Staining wasevaluated and MinFeret diameters of fibers were quantified using a NikonEclipse 90i microscope using NIS-Elements AR software (Nikon InstrumentsInc.). Immunofluorescence staining was performed as described above formouse muscle samples. For histological studies of canine tissuefollowing intravascular infusion of rAAV-MTM1, biopsies of thequadriceps muscles from the infused and non-infused limb were taken at14 and 27 weeks of life from 3 normal dogs and 3 treated XLMTM dogs.Sectioning and staining was using HE and NADH were performed asdescribed above. Fiber size, internal nucleation, and organellarlocalization were measured and quantified manually using an BX53microscope and CellSens Standard software (Olympus). The number offibers assessed ranged from 256 to 695.

For ultrastructural studies, a small portion of the mid-tibialisanterior muscle from 2 normal dogs and 2 XLMTM dogs was fixed in 5%glutaraldehyde, 2.5% paraformaldehyde in 0.2 mmol/L of cacodylatebuffer, pH 7.4. Fixed tissue was then subjected to osmication, stainedusing uranyl acetate, dehydrated in alcohol, and embedded in TAAB Epon(Merivac Ltd.). Subsequently, 1 μm scout sections were evaluated andareas of interest were cut at 95 nm thickness using an ultracutmicrotome (Leica Camera AG), picked up on 100 m formvar-coated coppergrids, stained with 0.2% lead citrate, and post-stained with uranylacetate to improve resolution of the transverse (T) and (L) longitudinaltubules of the sarcotubular system. Tissue was viewed and imaged using aTECNAI™ BioTwin Spirit Electron Microscope (FEI Co.). The number of Tand L tubules was quantified manually by evaluation of onerepresentative 6800× image from each myofiber within 1 or 2well-oriented specimens. The number of myofibers quantified ranged from6-11 fibers per specimen for WT animals and 7-12 myofibers per specimenfor XLMTM dogs.

Computerized Tomography (CT) Analysis of Canine Muscle

Digital data from anesthetized affected male XLMTM dogs (n=3) and normalmale littermates (n=3) were obtained from 0.5 mm serial whole-body CTimages (General Electric 8800) taken at the end of the study, just priorto necropsy. An investigator blinded to the experimental interventiontraced the area of interest in each image using a digital pen (Cintiq,Wacom). Image settings were kept consistent to minimize variance amongtracings. Digital area and volume measurements (Li et al., 2008, VetParasitol 157(1-2):50-8) of muscles were obtained as described (Wang etal., 2008, Gene Ther 15(15):1099-106) using OsiriX, an open-sourcesoftware (Bretag, 2007, Nature 450(7173):E23; discussion E23-5).

Immune Response Profile of Canines Following rAAV8-MTM1

Humoral Immune Response to Vector:

Enzyme-linked immunosorbent assay (ELISA) was used to detect the IgG andIgM antibodies specific to AAV8 vector in sera of dogs, as previouslydescribed for humans (Boutin et al., 2010, Hum Gene Ther 21(6):704-12;Monteilhet et al., 2011, Mol Ther 19(11):2084-91). These ELISA wererevealed by a colorimetric amplification system based on alkalinephosphatase and results expressed as titer. A neutralizing assay wasemployed to detect neutralizing factors (NAF) specific to AAV8 in dogsera, as previously described for human sera (Boutin et al., 2010, HumGene Ther 21(6):704-12; Monteilhet et al., 2011, Mol Ther19(11):2084-91). Briefly, vectors and serum were mixed under appropriateconditions and inoculated into cell culture susceptible to the vector.The loss of infectivity was brought about by interference by the boundantibody with any one of the steps leading to the release of the viralgenome into the host cells. The values are presented as percentage oftransgene activity compared to transduction of an equal amount ofvectors without serum pre-incubation. The neutralizing titer wasdetermined as the serum dilution at which 50% or higher inhibitionoccurred.

Humoral Immune Response to MTM′ Transgene:

Customized ELISA assays were developed to detect the IgG and IgMantibodies specific of MTM1 protein in sera of animals. For that,purified canine MTM1 protein was used. These ELISA were revealed by acolorimetric amplification system based on alkaline phosphatase andresults were expressed as titer.

Cellular Immune Response to AAV Vectors:

Cellular response specific to AAV in dog was by IFNγ ELIspot assays, aspreviously described for humans (Herson et al., 2012, Brain 135(Pt2):483-92). Briefly, frozen peripheral blood mononuclear cells (PBMC)were plated into an IFNγ precoated 96-well ELIspot plate, and incubatedin the presence of lentiviral vectors coding for VP1, VP2 and VP3 capsidproteins of AAV (LV-Cap8). Results were expressed as spot-formingunits/10⁶ cells. Samples were considered positive for AAV if the numberof spots was greater than 1.8 times the corresponding control withLV-empty.

Cellular Immune Response to MTM′ Transgene:

Cellular response specific to MTM1 was measured by ELIspot assays indogs. Frozen PBMCs were plated into an IFNγ precoated 96-well ELIspotplate, and incubated in the presence of lentiviral vectors coding forthe whole MTM1 protein (LV-MTM1). Results were expressed as spot-formingunits/10⁶ cells. Samples were considered positive for MTM1 if the numberof spots was greater than 1.8 times the corresponding control withLV-empty.

Inflammatory Immune Response:

Quantification for IL2, IL6, IL8, IL10, IL15, IFNγ and TNFα wasperformed at various time points pre- and post-vector administration, byLuminex technology as described (Skogstrand, 2012, Methods 56(2):204-12;Malekzadeh et al., 2012, Methods 56(4):508-13).

Muscle Function

Actimeter Test

Spontaneous locomotor activity in mice was assessed using the LE 8811 IRmotor activity monitor (Bioseb). Briefly, mice were placed in an openfield bounded with 16 horizontal photoelectric Infra Red beams tomeasure three-dimensional movements of the animals. The distance crossedwas recorded and analyzed for 90 minutes.

Escape Test

Global strength of mice was evaluated by the “escape test” (Carlson andMakiejus, 1990, Muscle Nerve 13(6):480-4). Briefly, mice were placed ona platform facing the entrance of a 30 cm tube. A cuff wrapped aroundthe tail was connected to a fixed force transducer and the mice wereinduced to escape within the tube in the direction opposite from theforce transducer by a gentle pinching of the tail. The mouse's forwardflight induces a short peak of force. The average of the five highestforce peaks, normalized to body mass, was reported.

EDL Contractile Force in Mice

Measurements of isometric contractile properties of murine EDL muscleswere performed in vitro as described (Buj-Bello et al., 2008, Hum MolGenet 17(14):2132-43). Animals were anesthetized by intra-peritonealinjection of pentobarbital (50 mg/kg). The muscles were surgicallyexcised and soaked in an oxygenated Krebs solution maintained at 20° C.Muscles were connected at one end to an electromagnetic puller and atthe other end to a force transducer, and stimulation was deliveredthrough electrodes running parallel to the muscle. Twitch and tetanic(125 HZ, 300 ms) isometric contractions were recorded at L₀ (the lengthat which maximal tetanic isometric force is observed). For comparativepurposes, normalized isometric force was assessed. Isometric specificforce (tension) was calculated by dividing the force by the estimatedcross-sectional area of the muscle (Dubey et al., 2007, Vet Parasitol149(3-4):158-66).

Hind Limb Contraction in Dogs

Contractile properties in canine muscles in vivo were assessed asdescribed (Tegeler et al., 2010, Muscle Nerve. 42(1):130-2; Childers etal., 2011, J Vis Exp. 2011 Apr. 5; (50). pii: 2623). Briefly, the hindlimbs of anesthetized dogs were immobilized in a frame to align thetibia at a right angle to the femur. Hind limb torque was measured bywrapping the foot to a pedal mounted on the shaft of a servomotor thatalso functioned as a force transducer. Percutaneous stimulation of theperoneal nerve activated hind limb muscles to pull the foot up towardthe body to generate torque. Computer software controlled theservomotor, stimulation timing, and capture of torque responses.Isometric contractions were performed over a range of stimulationfrequencies to determine torque-frequency relationships (FIG. 4B). Thisprocedure was followed by a series of repeated contractions, firstinduced by an initial short isometric contraction followed by a forcedstretch (eccentric contraction) lasting less than 1 sec. Contractionswere repeated every 5 sec. for a total of 30 activations (FIGS.25A-25B). Contractile data were analyzed as described (Tegeler et al.,2010, Muscle Nerve. 42(1):130-2) by investigators blinded to theexperimental treatment.

Statistical Analysis

Statistical analyses were performed using SAS software (Version 6; SASInstitute Inc, Cary, N.C.). Individual means were compared usingnon-parametric tests (Mann-Whitney, or Wilcoxin rank-sum tests).Differences were considered to be statistically significant at eitherP<0.05 (1 symbol, *), or P<0.01 (2 symbols, **), or P<0.001 (3 symbols,***). All data are presented as means±Standard Error of the Mean (SEM).

The results of the experiments are now described

Systemic Mtm1 Delivery Improves Growth and Survival ofMyotubularin-Knockout Mice

Untreated myotubularin-knockout mice, whether constitutive (whole-body;KO-Mtm1) or muscle-specific (mKO-Mtm1), display muscle pathology by 3weeks-of-age (FIGS. 8A-8B) and survive, on average, less than 2 months(Buj-Bello et al., 2002, Proc Natl Acad Sci USA 99(23):15060-5).

To correct the MTM1 deficiency, two muscle-tropic AAV vectors serotype 8(AAV8) and serotype 9 (AAV9) were tested herein.

1) Use of Muscle-Tropic rAAV8 Vector in Mice:

For systemic delivery of Mtm1, the muscle-tropic serotype 8 AAV vectorwas developed expressing the Mtm1 complementary DNA (cDNA) under thecontrol of a muscle-specific desmin promoter (AAV2/8-pDesmin-Mtm1,abbreviated AAV8-Mtm1 or more generally rAAV-Mtm1). The vector wasproduced in human embryonic kidney (HEK) 293 cells by atritransfection-based system, formulated for in vivo injection, and usedto treat two groups of KO mice at different stages of disease evolution,at the early onset of the pathology (3 weeks of age) or at the latestage of the disease when mortality occurs (5 weeks of age) (FIG. 1A). Asingle tail vein injection of AAV8-Mtm1 at a dose of 3×10¹³ vg/kg inMtm1 KO mice at 3 weeks (KO Early; n=8) conferred long-term survival andnearly normal growth on 100% of the treated animals (FIG. 1B and Movie 1described below). The same dose was administered to severely affectedmice at 5 weeks (KO Late; n=11), when 20% of the animals had alreadydied. All treated mice remained viable and gained body mass over a6-month observation period, except for a single 5-week-old mouse thatdied 1 day after injection (FIG. 1B and FIG. 1C). Consistent with theirrobust appearance, skeletal muscles grew to normal size invector-injected Mtm1 KO mice. In both the early- and late-treatedcohorts, each of the seven individual muscles analyzed gained mass,reaching >70% of the mass of wild-type muscle at 6 months (FIG. 1D).Analysis of myotubularin expression by Western blotting in individualmuscles at sacrifice demonstrated that intravenous delivery of AAV8-Mtm1reconstituted efficient myotubularin synthesis in skeletal musclesthroughout the body. Myotubularin levels ranged between one and fivefoldgreater than wild-type values in most skeletal muscles. There was nomajor difference between the early and late treatment cohorts, exceptfor two peaks of >15-fold expression in the soleus muscle ofearly-treated KO mice and >40-fold expression in the tibialis anteriorof late-treated KO mice (FIG. 1E). Myotubularin was highly overexpressedin the heart (720±153 times the endogenous level 6 months aftertreatment) (FIG. 9C). The AAV8-Mtm1 average vector copy number (VCN;corresponding to viral genomes per diploid genome) in early andlate-treatedmice was 0.72±0.1 and 0.87±0.1 in the tibialis anteriormuscle and 1.67±0.4 and 3.33±2.1 in the biceps brachii muscle,respectively. VCN in the heart and liver ranged between 1.17 to 5.14 and80 to 223 viral genomes/diploid genome, respectively, consistent withthe known tropism of AAV8. At necropsy, the heart of AAV-treated KO miceshowed the presence of some focal lesions with scar tissue and a modestcellular infiltrate, although these lesions did not affect survival inany of the treated Mtm1 KO animals.

Movie 1, available at www dot sciencetranslationalmedicine dotorg/cgi/content/full/6/220/220ra10/DC1 in a mp4 format, shows in thesame cage a wild-type mouse (red tail and tag) injected with PBS and amyotubularin-deficient mouse (blue tail and tag) treated with AAV8-Mtm1(3×10¹³ vg/ml) at 6 months after injection. One can clearly note therobust appearance, normal size, and activity of the treated Mtm1 KOmouse which is very similar to the wild-type mouse.

2) Use of Muscle-Tropic rAAV9 Vector in Mice:

For systemic delivery of Mtm1, a muscle-tropic rAAV9 vector was used andthe desmin promoter was employed to restrict expression to muscle(rAAV2/9-pDesmin-Mtm1^(murine), abbreviated AAV9-Mtm1 or more generallyrAAV-Mtm1). It was observed that in both strains a single intravenousinjection of vector conferred long-term survival and nearly normalgrowth (FIGS. 19A-19H). When 5 symptomatic KO-Mtm1 mice receivedrAAV-Mtm1 (3.0×10¹³ vg/kg), all remained viable and gained body massover a 6-month observation period (FIG. 19A and FIG. 19B). The study wasreplicated in 6 mKO-Mtm1 mice using a lower vector dose (0.5×10¹³vg/kg). In this case, 5 treated animals survived and grew normallyduring a 12-month follow-up, and the sixth died well beyond the usualsurvival limit (FIG. 19E and FIG. 19F). Consistent with their robustappearance and activity, the vector-injected Mtm1-defective miceachieved gross normalization of their skeletal muscles. In the treatedKO-Mtm1 cohort, every individual muscle assessed gained mass by thesecond week post-injection, and muscle masses reached >80% of WT at 6months (FIG. 19C). Similarly, the muscle masses of vector-treatedmKO-Mtm1 mice exceeded 75% of WT at 12 months (FIG. 19G).

Immunoblotting demonstrated that intravenous delivery of rAAV-Mtm1enabled efficient, sustained production of myotubularin in skeletalmuscles throughout the body. In the KO-Mtm1 mice at 2 weekspost-injection, the relative level of myotubularin in most individualmuscles exceeded WT muscles by at least 5-fold (FIG. 19D). At 6 months,myotubularin expression ranged between 7- to 40-fold greater than WT.The muscles of treated mKO-Mtm1 mice, which received one-sixth thevector dose, were also uniformly positive for myotubularin protein. At12 months post-injection, the levels were near normal—approximately 30%of WT in the diaphragm, and 60% to 140% in various other muscles (FIG.19H).

Mtm1 Gene Therapy Corrects the Pathology of Myotubularin-Deficient MouseMuscles

The muscles of myotubularin-knockout mice that received a muscle-tropicAAV vector expressing Mtm1 gene underwent rapid and sustainedamelioration of pathological features.

FIGS. 2A-2B show representative histology from the tibialis anterior andbiceps brachii limb muscles of untreated, 5-week-old Mtm1 KO mice andAAV-treated, 6-month-old KO mice, demonstrating normalizedcross-sectional fiber size and intracellular architecture, as revealedby hematoxylin and eosin (H&E) and NADH (reduced form of nicotinamideadenine dinucleotide) tetrazolium reductase (NADH-TR) staining (seeFIGS. 9A-9C for additional information). Morphometry of tibialisanterior and biceps brachii myofibers from 5-week-old Mtm1 KO mice gavea mean diameter of 15.9±0.8 mm and 17±0.7 mm, respectively, with manyfibers below 20 mm, compared to 28.7±0.8 mm and 24.3±1.2 mm forwild-type mice (FIG. 2B and FIG. 9A). Six months after treatment withAAV8-Mtm1, the abundance of extremely small-diameter myofibers waseliminated, and the size distribution approached that of wild-typemuscles in both cohorts. In addition, myofibers of treated micedisplayed a reduced frequency of centrally localized nuclei, adiagnostic feature of centronuclear myopathies like XLMTM (FIG. 9B).Distinctive features of myotubularin-deficient myofibers includeaberrant accumulations of mitochondria and a marked deficiency oftransverse tubules (T-tubules), invaginations of the plasma membraneperpendicular to the length of the myofiber that are critical forexcitation contraction coupling and muscle function. Untreated Mtm1 KOmice showed abnormal localization of proteins associated with theT-tubule system, including the dihydropyridine 1a receptor (DHPR1a), avoltage-gated Ca²⁺ channel, and dysferlin, a transmembrane proteininvolved in Ca²⁺-dependent membrane repair (FIG. 2A, arrows). Treatmentwith AAV8-Mtm1 corrected abnormal mitochondria distribution and cellularmislocalization of DHPR1a and dysferlin in all mice from both early- andlate-treated cohorts, indicating that XLMTM-associated pathology can bereversed well after the onset of the disease.

FIG. 20A-20F show representative data from the tibialis anterior (TA)muscle of KO-Mtm1 animals at 2 weeks and at 6 months after the singlesystemic vector treatment. (See FIGS. 22A-22B for additional muscles).The cross-sectional area of limb muscles enlarged (FIG. 20A) and musclefibers increased in size (FIG. 20B, FIG. 20F FIG. 22A and FIG. 22B).Morphometry of untreated mutant TA myofibers at 5 weeks-of-age gave amean diameter of 15.9±1.7 μm, with many below 20 μm, compared to28.7±2.1 μm for WT (FIG. 20F). By 2 weeks post-treatment with rAAV-Mtm1,the skew towards extremely small diameter myofibers was eliminated, andat 6 months the size distribution approached that of WT. In addition,myofibers of treated KO-Mtm1 and mKO-Mtm1 mice displayed a reducedfrequency of internalized nuclei, a diagnostic feature of centronuclearmyopathies such as XLMTM (FIG. 23A).

Mtm1 gene delivery also corrected the intracellular muscle architectureof the mutant mice, as revealed by hematoxylin-eosin andNADH-tetrazolium reductase (NADH-TR) staining (FIG. 20B and FIG. 20C).Distinctive features of myotubularin-deficient myofibers includeaberrant accumulations of glycogen and mitochondria, and a markeddeficiency of transverse tubules (T-tubules)—invaginations of the plasmamembrane perpendicular to the length of the myofiber critical forexcitation-contraction coupling and, thereby, muscular function.Immunofluorescent staining of muscle from KO-Mtm1 mice showed abnormallocalization of proteins associated with the T-tubule system, includingdihydropyridine 1α receptors (DHPR1α; voltage-gated calcium channels)and dysferlin, a transmembrane protein involved in calcium-dependentmembrane repair (FIG. 20D and FIG. 20E). Consistent with the previousobservations after intramuscular vector delivery (Buj-Bello et al.,2008, Hum Mol Genet 17(14):2132-43), systemic myotubularin gene therapyrapidly restored normal myofiber morphology, in particular the cellularlocalization of DHPR1α and dysferlin. This restoration was maintainedthrough the 6-month assessment. Myofiber ultrastructure inrAAV-Mtm1-treated mutants appeared normal by electron microscopy, withproperly distributed T-tubules and triads (FIG. 24).

Structural abnormalities of muscle in Mtm1-defective mice are mirroredby functional deficits. Whole-body and muscle-specific knockouts displayequivalent phenotypes, implying that myotubularin deficiency in skeletalmuscle is necessary and sufficient to account for the dysfunction.

Structural muscle abnormalities are mirrored by severe functionaldeficits in Mtm1 KO mice. To measure the effect of gene therapy onmuscle function, the open-field actimeter, global muscle strength, andisolated limb strength assays were used. Open-field actimetermeasurements showed that mutant mice covered less than half the distanceexplored by wild-type mice at 5 weeks of age (FIG. 3A). Mice treatedwith AAV8-F3 Mtm1 at both early and late stages of the disease showedsignificant functional improvement, and at 6 months after AAV injection,their motor activity was indistinguishable from that of wild-typeanimals. A noninvasive test of global muscle strength that measuresforward pulling tension in an escape paradigm revealed that untreatedMtm1-deficient mice were half as strong as wild-type mice (whole-bodytension, 0.07±0.01 versus 0.15±0.01 N/g; P<0.01) (FIG. 3B). Early- andlate-treated mice showed 82% (0.15±0.01N/g) and 76%(0.13±0.01N/g)recovery of whole-body tension, respectively (FIG. 3B). In a functionalassay of an isolated hindlimb muscle, the extensor digitorum longus, theisometric force of untreated Mtm1 KO mice was only 13% of the wild-typelevel, whereas it almost normalized 6 months after AAV8-Mtm1 delivery inboth cohorts (P=0.0016 and P<0.001 for the early and late-treated groupsof mice, respectively; FIG. 3C). Together, these data indicate thatmuscle impairment associated with myotubularin deficiency can be rescuedby gene therapy even after the onset of pathology.

Multiple tests demonstrated long-term restoration of muscle function bysystemic myotubularin gene therapy in KO-Mtm1 (FIG. 3) and mKO-Mtm1 mice(FIG. 23B and FIG. 23C). Open-field actimeter measurements demonstratedmutants at 5 weeks-of-age displayed fewer than half the episodes ofspontaneous motor activity, and covered less than half the distanceexplored by WT mice (FIG. 20A). However, KO-Mtm1 mice that receivedsystemic rAAV-Mtm1 2 weeks prior to the test showed significantimprovements. At 6 months post-therapy (or 12 months in the mKO-Mtm1study, FIG. 23B) their behavior was indistinguishable from WT. Anothernoninvasive test of motor activity and global strength, initiallyvalidated in a mouse muscular dystrophy model, measured forward pullingtension in an escape paradigm (FIG. 20B). By this assay, 5 week-oldKO-Mtm1 mice were approximately half as strong as WT (whole body tension0.07±0.01 versus 0.15±0.01 mN/g). The improvement in mutants given genetherapy 2 weeks earlier was pronounced; their whole body tension was 87%of normal (0.13±0.01 mN/g). After 6 months, the treated mutants stillperformed similarly to WT.

Finally, the function of an isolated hind limb muscle, the extensordigitorum longus (EDL) was assessed in an isometric force assay (FIG. 3Cand FIG. 23C). EDL muscle strength of untreated KO-Mtm1 mice was only13% of WT. Two weeks after rAAV-Mtm1 delivery, the strength of mutantmuscles increased nearly 4-fold over saline-injected controls. By 6months, the strength of EDL muscles from vector-treated mutants equaledthat of WT.

Correction of Muscle Pathology in Myotubularin-Deficient Mice isDose-Dependent

To assess the effect of a lower vector dose on phenotype correction,AAV8-Mtm1 (5×10¹² vg/kg) was injected into the tail vein of Mtm1 KO miceat 3 weeks of age (n=10). This dose prolonged the survival of 50% of themice over 3 months, with the first death occurring 6 weeks afterinjection (FIG. 10A). Body weight increased during the first 3 weeks oftreatment and remained unchanged after this period (FIG. 10B). Thispartial recovery of body mass was reflected at the level of individualskeletal muscles: two of the seven analyzed muscles (soleus and bicepsbrachii) grew normally, whereas the other five reached 40 to 60% ofwild-type mass at 3 months (FIG. 10C). Themotor activity of mice treatedwith the low vector dose appeared indistinguishable from that of wildtype (WT) mice and KO mice treated with the high dose in an open-fieldactimeter assay 3 months after injection (FIG. 10E). However, theirglobal muscle strength was reduced by 55% in the more sensitive escapetest, and their isolated soleus and extensor digitorum longus musclesgenerated 60 and 9% of the wild-type force, respectively, indicatingthat muscle function recovery was not complete in the low-dose cohort.In the extensor digitorum longus muscle, only 3% of the myotubularinendogenous level was reached (FIG. 10D), ranging from 7 to 27% in theother analyzed muscles (mean=13%), indicating that low levels of MTM1are sufficient to prolong the survival of mutant mice.

rAAV8-MTM1 Administration Rescues Pathology of XLMTM Canine Muscles

To evaluate myotubularin gene replacement therapy in a large animalXLMTM model, affected male Labrador/beagle F1 offspring, which displaythe same pathology and clinical features as previously reported foraffected purebred Labradors, were studied. The mutant dogs becomesymptomatic by 9 to 10 weeks-of-age. Muscular weakness progresses forapproximately 6-8 more weeks to ˜18 weeks-of-age, when animals can nolonger ambulate and experiments are terminated.

Effects of Intramuscular Delivery of rAAV8-MTM1.

The first goal was to confirm transgene expression in skeletal muscle,using the muscle-tropic serotype 8 AAV vector to deliver canine MTM1cDNA driven by the desmin promoter. A single intramuscular injection(4×10¹¹ vg/kg) of rAAV8-pDesmin-MTM1^(canine) (rAAV-MTM1) wasadministered into a hind limb, at the middle of the cranial tibialismuscle, of each of three XLMTM dogs at 10 weeks-of-age. Thecontralateral limb received only saline. Saline-injected unaffected malelittermates served as WT controls. Immunoblotting confirmed substantialamounts of myotubularin protein in rAAV-MTM1-injected muscles of all 3XLMTM dogs (FIG. 21A). After 4 to 6 weeks, the level relative to WT wasapproximately 60% near the center of the cranial tibialis and about 8%at its ends (FIG. 21B). Some transgene-encoded protein was also observedin the contiguous EDL muscle. However, neither the contralateral limbmuscles, nor distant skeletal muscles such as diaphragm, nor heartmuscles contained detectable myotubularin.

In every case, the mutant muscle injected with rAAV8-MTM1 grewsignificantly larger than the contralateral control muscle (FIG. 4A andFIGS. 11A-11B). Weight measurements and computed tomography (CT) scansshowed that myotubularin gene therapy increased muscle mass (FIG. 21Cand FIG. 21D) and volume by about 50% in 4 to 6 weeks (P=0.079) (FIGS.11A-11B), respectively.

Skeletal muscles from myotubularin-deficient dogs, similar to those frommyotubularin-knockout mice, display histological aberrations typical ofXLMTM. Muscles of rAAV-MTM1-injected mutant canine limbs consistentlyshowed improved architecture, increased myofiber size, and lessmislocalization of organelles (including mitochondrial aggregates and“necklace” fibers) (FIG. 4C; Table 1). Myotubularin gene therapy alsocorrected the localization of DHPR1α and dysferlin as observed byimmunofluorescent staining (FIGS. 4A-4C). Electron microscopy confirmedthat, by comparison with WT muscle, XLMTM muscle contained fewcharacteristic T-tubules but showed atypical longitudinal structures(L-tubules). By contrast, rAAV-MTM1-injected mutant muscles closelyresembled WT, with abundant T-tubules (FIG. 4C; Table 1 listed below).

TABLE 1 Quantified Histological Findings in Dogs Following IntramuscularInjection XLMTM + XLMTM + WT + saline saline AAV p Value HistologicalFindings Mean MinFeret 32.0 ± 8.0  17.3 ± 4.1  30.2 ± 3.7  0.06 FiberDiameter (μm) % Fibers with Central 0 7.5 ± 5.1 5.4 ± 4.0 0.42 Nuclei %Fibers with Central 0 17.1 ± 4.0  4.3 ± 6.6 0.14 Organelle Aggregates %Necklace Fibers 0 3.19 ± 16.8   4 ± 6.7 0.17 Ultrastructural Findings TTubules/Fiber 10.6 ± 1.5  3.9 ± 2.0 10.2 ± 3.0  0.04 L Tubules/Fiber0.18 ± 0.01 1.56 ± 0.20 0.18 ± 0.25 0.15

Effects of Intravascular Delivery of rAAV8-MTM1.

In other experiments, a single intravascular infusion of rAAV8-MTM1(2.5×10¹² vg/kg) was administered into the hindlimb saphenous vein underhigh pressure as described in each of three XLMTM dogs at 9weeks-of-age. The contralateral limb was not infused. At 13 and 17weeks-of-age muscle biopsies were obtained from hind and forelimbs, andmuscle lysates probed with anti-myotubularin antibodies. Immunoblotsdemonstrated myotubularin transgene expression in all muscle samplestaken from XLMTM dogs (FIGS. 25A-25B): four weeks after rAAV8-MTM1infusions, the level of myotubularin expression in treated dogs(relative to WT expression) was increased. Infusion with rAAV8-MTM1 alsoshowed improved myofiber architecture and increased myofiber size (FIGS.26A-26C). Specimens taken from the infused limb showed a consistentfiber size and appearance, with a smaller average fiber size than isseen in WT muscle. In contrast, the non-infused limb displayed markedvariation in fiber size with populations of very small and very largefibers, and two of the three animals displayed a larger average fibersize than was seen in the WT animals. The single biopsy from anon-infused limb that showed smaller fiber size than WT animals wastaken adjacent to a myotendinous insertion site, which is known tolocally affect fiber size and may not be characteristic of the majorityof this muscle. Mislocalization of organelles was only seen in one ofthe three rAAV8-MTM1-infused dogs and was higher in the non-infused limb(FIGS. 6A-6B). Electron microscopy performed on quadriceps musclesamples from infused and non-infused limbs at 4 and 8 weekspost-treatment displayed appropriate sarcotubular organization, similarto what was seen following intramuscular injection. Electron microscopyof the biceps femoris muscle at the terminal time point revealed adecrease in sarcotubular organization in both the infused andnon-infused hindlimb, with only approximately 1 triad seen per field ineach muscle.

MTM1 Replacement Improves Strength of XLMTM Canine Muscle

To measure function of the treated limbs, force transduction assays,originally developed to study canine muscular dystrophy, were used. FIG.4B depicts an isometric contraction assay. Briefly, non-invasiveelectrical nerve stimulation, of differing frequencies, at the stiflejoint (knee) of an anesthetized dog stimulates muscle contraction andfoot flexion analogous to lifting off the ground during walking. Thehind foot is affixed to a pedal mounted on the shaft of a forcetransducer so that measured torque reflects the strength of the lowerlimb muscles. Immediately prior to gene therapy at 10 weeks-of-age(baseline), XLMTM dogs were mildly weaker than unaffected WTlittermates, and the two hind limbs of each dog performed equally (FIG.4B). Measurements 4 and 6 weeks later showed that the limb strength ofWT dogs more than doubled, consistent with normal muscular maturation(FIG. 4B and FIG. 5B). However, in XLMTM dogs, limbs injected withsaline intramuscularly did not improve, so that the strength deficitrelative to WT became worse.

Strength Improved after Intramuscular Administration of rAAV8-MTM1.

Each mutant limb injected with rAAV8-MTM1 strengthened significantly by14 weeks-of-age (FIG. 4B). In the 2 dogs maintained until 16weeks-of-age, the treated limbs continued to improve, reaching 80% of WTtorque versus 30% for untreated limbs (FIG. 5B).

A dynamic eccentric contraction assay was also used to measure muscularperformance during repetitive exercise. (FIG. 12A). When assessed bythis test over time (i.e. age), the hind limbs of young WT dogsstrengthened, while intramuscular saline-injected limbs of the threeXLMTM dogs declined to about 20% relative to WT. Again, local rAAV8-MTM1therapy greatly improved exercise performance; at 14 and 16weeks-of-age, treated limbs achieved >70% of WT strength (FIG. 12Bthrough FIG. 12D).

These results showed that a single injection of AAV8-MTM1 is efficaciousin rescuing the function of an entire myotubularin-deficient muscle,prompting the assessment of an intravascular delivery approach.

Intravascular Administration of AAV8-MTM1 Rescues Muscle Pathology andProlongs Survival of XLMTM Dogs.

AAV delivery by isolated limb perfusion allows widespread transductionof muscle groups in dogs and nonhuman primates. To test whether regionaladministration is sufficient to ameliorate muscle pathology in an entirelimb, a single dose of AAV8-MTM1 (2.5×10¹³ vg/kg) was injected underhigh pressure into the saphenous vein of three 9-week-old XLMTM dogs,after applying a tourniquet around the hindlimb distal to the injectionsite to limit blood circulation during infusion. The tourniquet wasreleased 5 min after injection. Treated dogs improved in strengthrapidly after vector administration. In contrast to dogs injectedintramuscularly, where only the muscles of the injected limbgainedstrength, high-pressure intravascular limb delivery of AAV8-MTM1resulted in improved strength of both the infused and contralateralhindlimbs, which reached on average almost normal values 6 weeks afterinfusion (FIG. 5A and FIGS. 13A-13C). Similarly, peak inspiratory flow(PIF) (the fastest flow rate measured during inhalation) was stronglyreduced in XLMTM dogs at 16 weeks of age, but was normal in treated dogs(FIG. 5B), indicating that the AAV8-MTM1 vector transduced respiratorymuscles and improved their function. Most importantly, all treated dogsshowed a marked improvement in survival, which extended far beyond thecritical 18-week time point, when all untreated XLMTM dogs could nolonger ambulate and needed to be euthanized. The first infused XLMTM dog(dog 4) survived in relatively good condition beyond 1 year and wassacrificed for analysis at 14 months of age. The other two dogs (dogs 5and 6) remained ambulant and clinically robust beyond the age of 1 year,and were alive and healthy at the time of disclosure preparation (Movie2, described below). Muscle biopsies were taken from the injected andcontralateral hindlimbs (quadriceps and biceps femoris) and from theforelimbs (triceps and biceps brachii) of all treated dogs 4 weeks afterinfusion to analyze vector distribution, transgene expression, andhistology. Intravenous administration of AAV8-MTM1 improved myofiberappearance and architecture in the treated limbs, which consistentlyshowed an average fiber size only slightly reduced compared to wild-typemuscles measured 4 weeks after infusion (FIG. 6A). In contrast, at 1year after infusion, noninfused limbs displayed heterogeneity in musclefiber size, with coexistence of small and large fibers (FIG. 6B).Electron microscopy performed on quadriceps muscle samples obtained fromall infused limbs showed normalized sarcotubular organization, similarto what was observed after intramuscular injection (Table 2, listedbelow). The biceps femoris muscles of dog 4, analyzed at sacrifice,revealed a partially defective sarcotubular organization in both theinfused and non infused hindlimb, with only about one triad per field ineach muscle (Table 3, listed below). Real-time polymerase chain reaction(PCR) and Western blot analysis demonstrated both AAV8 vector (FIG. 7A)and myotubularin (FIG. 7B and FIG. 14) expression in all muscle samplesobtained from all three treated dogs: 4 weeks after infusions, the levelof myotubularin expression reached 21 to 72% of the wild-type controllevels in the biceps femoris and quadriceps muscles in the injectedlimbs and 9 to 138% in the muscles (quadriceps, biceps femoris, triceps,and biceps brachii) of the non injected limbs. Expression levels wereparalleled by VCN levels, which ranged from 0.52 to 2.35 in the bicepsfemoris and quadriceps muscles of the infused limbs and from 0.17 to3.65 in the muscles of the non injected limbs (FIG. 7A). Thebiodistribution of AAV8-MTM1 was analyzed in dog 4 at necropsy. The VCNwas, in general, higher in infused muscles (range, 0.01 to 2.27) than indistal muscles (range, 0.007 to 0.13; n=18), with the exception of thediaphragm, intercostal muscles, and heart, where it was detected a VCNof 0.37, 0.59, and 0.28, respectively (FIG. 7C). MTM1 protein levelswere above the endogenous level in 7 of 13 muscles from the infusedhindlimb and barely detectable in the contralateral and forelimb muscles(FIG. 7D), mirroring the VCN values and suggesting that very low amountsof myotubularin are sufficient to rescue muscle function. In thediaphragm and heart, myotubularin reached 64 and 13% of the wild-typevalues, respectively. The heart of dog 4 showed no signs of toxicity athistological examination (FIG. 15) and at the functional level, asassessed by electrocardiogram and echocardiography before necropsy. Thehistology of the liver (VCN: 0.63) was also normal, and myotubularinprotein was undetectable. These data show that isolated limb perfusionwas effective in delivering the AAV vector to the infused muscle groupsbut did not limit vector diffusion to other organs, including the restof the skeletal musculature and the heart.

Movie 2, available at www dot sciencetranslationalmedicine dotorg/cgi/content/full/6/220/220ra10/DC1 in a m4v format, comprises eightsections that illustrate a normal healthy control dog, two untreatedXLMTM dogs and three XLMTM dogs (Dog 4, Dog 5 and Dog 6) treated withAAV8-MTM at different ages.

-   Section 1: A normal healthy dog is shown along with a XLMTM affected    dog. The normal dog is moving very actively and trying to get a    treat from the trainer's hand. The affected dog is lying on the    floor with difficulties to ambulate and to stand for reaching toward    the treat in the trainer's hand. After few trials, the healthy dog    successfully gets the treat while the affected dog was clearly too    weak to compete against him.-   Section 2: An untreated dog, at 4 months of age, is lying on the    floor having clear difficulties to ambulate.-   Section 3: Another untreated dog, at 4 months of age, is shown a can    of food by a trainer. The dog smells the can several times but does    not seem interested in eating it.-   Section 4: Dog 4, at 4 months of age, is seen very active and    playing with a towel.-   Section 5: Dogs 5 and 6, both at 6 months of age, are seen very    active playing and jumping on each other.-   Section 6: Dog 4 is seen again at 1 year of age, on a leash walking    actively back and forth with his trainer then standing up to reach    toward a piece of meat shown to him by the trainer.-   Section 7: Dogs 5 and 6, both at 1 year of age post infusion, are    seen playing very actively with a towel and chasing each others    through a gymnastic lap that includes a tunnel and a step.-   Section 8: Dogs 5 and 6, both at 1 year of age post infusion, are    seen playing very actively and trying to steal a towel from each    other's muzzle.

TABLE 2 Quantified Histological Findings in Dogs after Intravenous AAV.Mean MinFeret % Fibers with Fiber Diameter % Fibers with Mislocalized(μm) Internal Nuclei Organelles Control (WT) Animals Dog C1 31.0(8.7-54.9) 0 0 Dog C2 31.1 (4.9-68.8) 0.5 0 Dog C3 26.2 (6.2-44.9) 0.2 0Experimental Animals Dog 4 Infused Leg 24.8 (3.6-65.0) 1.7 5.6Non-infused Leg 32.7 (6.0-65.8) 6.3 34.1 Dog 5 Infused Leg 26.2(5.5-61.4) 0.6 0 Non-infused Leg 37.7 (6.3-73.8) 0.8 0 Dog 6 Infused Leg22.7 (2.9-53.1) 0.9 0 Non-infused Leg 21.7 (3.1-71.2) 2.0 0

TABLE 3 Quantified Histological Findings in Autopsy Tissue at 1 yearafter infusion. Mean % Fibers % Fibers MinFeret with with Fiber DiameterInternal Mislocalized Muscle (μm) Nuclei Organelles Dog 4 InfusedCraniotibialis 38.7 (6.2-97.8)  1.3 0.3 Non-infused Crantiotibialis 30.8(4.7-137)   9.2 17.3 Infused Vastus Lateralis 30.0 (3.2-96.1)  7.3 11.3Non-infused Vastus Lateralis 54.1 (4.7-147.1) 4.0 3.3Immune Response Profile in XLMTM Dogs Following rAAV8-MTM1 HumoralImmune Response to Vector

NAF, IgG and IgM titers measured from the sera before and afterintramuscular (FIG. 16A) or intravenous regional limb (FIG. 16B)administration of rAAV8-MTM1 demonstrate that NAF and IgG titersincreased one week following injections and remained elevated for up to10 months. IgM titers decreased to pre-infusion baseline levels in XLMTMdogs given rAAV8-MTM1 by regional infusion.

Humoral Immune Response to MTM1 Transgene.

Compared to pre-infusion levels, elevations in IgG and IgM antibodiesspecific of MTM1 protein in sera were not observed in any XLMTM dogsgiven either intramuscular or intravenous infusions of rAAV8-MTM1 (FIG.16C and FIG. 16D).

Cellular Immune Response to AAV Vectors or MTM1 Transgene.

Cell-mediated immune responses against the vector or the transgeneproduct were tested by an interferon-g (IFN-g) enzyme-linked immunospotassay on peripheral blood mononuclear cells over a period of 155 daysafter vector administration. We were unable to detect any T cellsspecific to the vector capsid or the MTM1 protein in XLMTM dogs givenintramuscular or intravenous AAV8-MTM1 (FIG. 17 and FIG. 18).

Inflammatory Immune Response.

Innate immune response profiles of cytokines IL2, IL6, IL8, IL10, IL15,TNF-α and IFN-γ before and after intramuscular or regional limb infusionwith rAAV8-MTM1 are presented in Table 4 below. Of the three dogs, dog1, dog 2 and dog 3, given intramuscular rAAV8-MTM1, dog 3 displayedelevated TNF-α levels (42.5-80.8 pg/ml) between Day 3 to Day 28post-infusion. Notable elevated levels of innate responses were notobserved in other animals, including dog C, an untreated XLMTM controldog. Following regional rAAV8-MTM1 infusion, transient elevations in IL2and IL15 (743.5 and 119 pg/ml, respectively) were observed in one dog,dog 6, 6 hrs after infusion.

TABLE 4 Innate immune responses after intramuscular or regional limbadministration of AAV8- MTM1 in XLMTM dogs (dogs 1 to 6) and anuntreated XLMTM dog (dog C). IL2 IL6 IL8 IL10 IL15 TNF-α IFN-γ DogTiming (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) I.M Dog CPre-infusion 110.5 103.8 1587 29 63.5 86.8 9.5 injection D3  97 91 819.328 60.8 74.8 9.5 D14 84.5 74 1797 29 57.8 68.5 9.5 D21 81.6 80 886.829.5 52.8 60 9.5 D28 91.5 98.3 664.8 22 89 73.3 9 Dog 1 Pre-infusion 1923 255 26.3 30.5 33.5 7 D1  18.6 21.8 4417.5 33 20.8 28 7 D3  20.3 234257.9 35 27 36.5 6.5 D7  19.3 23 3393 33.5 27.5 37 6.5 D14 19.6 23.52616.9 33 26.5 36 6 D21 16 19.5 4076.4 36 22.5 29.5 7.3 D28 17.8 19.33375.9 35 21 28.5 7.5 Dog 2 Pre-infusion 16.5 21.5 3255.9 33 21 28 6.8D1  13 17.5 3907.5 29 18.5 23 6.3 D3  13.6 18 2709 33.5 18 25.8 7.5 D7 15.8 22.3 4977 27.5 26 29 6.5 D14 13.5 15 3115.5 32.5 19 23 6.5 D21 2024 2376 32.5 33 34.6 8.3 Dog 3 Pre-infusion 12.8 13 2472.9 27.5 15.519.5 7.3 D1  13 16 4586.4 32.8 16 21 7.5 D3  21.8 28 405 35.5 26.5 42.55.5 D7  20 27.5 803.4 31.5 23.5 44.3 5.8 D14 21.8 27.5 832.5 33.3 25.855.8 5 D21 22.3 31 283.5 34 28.5 80.8 6 D28 18.5 24.5 565.5 33.8 21.354.5 6 W7 12.5 17.5 2070 23.3 18.5 22.3 6.3 Regional Dog C Pre-infusion110.5 103.8 1587 29 63.5 86.8 9.5 infusion D3  97 91 819.3 28 60.8 74.89.5 D14 84.5 74 1797 29 57.8 88.5 9.5 D21 81.6 80 886.8 29.5 52.8 60 9.5D28 91.5 98.3 664.8 22 89 73.3 9 Dog 4 Pre-infusion 18 23.8 4865.3 4632.8 32.5 10.3 D0 + 6 h na na na na na na na D1  16 21.5 3415.6 40 3131.5 10.5 D3  18.3 23.3 4747.5 41 32 35.5 10.5 D7  23.5 32.5 2157.3 43.334.5 45.8 10 D14 15 18 566.5 46.5 28 28 10 Dog 5 Pre-infusion 68 113.5180.3 27 83.8 143 11 D0 + 6 h 81 143.5 744.5 33.5 103 202 10 D1  117.5125.8 304 34 93.3 197 10 D2  65.8 112 1310.3 34 84 148.5 10.3 D7  68.5113.5 689.3 34.5 72.5 139.5 11.3 D13 49.3 34 1665 29.5 45.8 20.5 8.5 Dog6 Pre-infusion 23.5 22.5 255.5 29.5 23.5 30.5 12.5 D0 + 6 h 743.5 20.5445 31 119 46.8 13 D1  14.8 15.6 303.5 34 13.5 19.8 12 D2  15 19 993.536 14.5 20.8 12.5 D7  12.8 15 170 35 13.5 20.8 11 D13 14 20.8 988 34.516 20.5 16.5 na: not applicableSystemic Mtm1 Delivery Improves Growth and Survival ofMyotubularin-Knockout Mice

FIGS. 1A-1E demonstrate that intravascular delivery of rAAV-Mtm1 inmyotubularin-deficient mice improves lifespan and body growth.Myotubularin-deficient mice were injected with rAAV-Mtm1 at 3×10¹³ viralgenomes per kg (vg/kg) at 3 (KO+AAV, n=12) and 5 weeks of age (KOLate+AAV, n=12) during a 6 months follow-up study. Both survival (FIG.1A) and body weight (FIG. 1B) is improved by treatment. FIG. 1C depictsthe mass of representative skeletal muscles of KO-Mtm1 mice 2 weeksafter injection of saline (KO+saline, n=4) and 6 months (n=10 afterinjection of rAAV-Mtm1 (KO+AAV, n=7, and KO Late+AAV, n=8). Values werenormalized to muscle mass of age-matched, saline-injected WT mice(n=10), taken as 100%. This data again demonstrates that treatmentincreases muscle size. Similarly, myotubularin protein quantification byimmunoblot compared to endogenous levels (line=1), demonstrates thattreatment increased myotubularin content.

FIGS. 2A-2B demonstrate that Mtm1 gene replacement therapy corrects theinternal architecture and hypotrophy of skeletal muscle fibers inmyotubularin-knockout mice. Mice were injected with either saline(+saline) or rAAV-Mtm1 vector (+AAV). Sections were obtained after 2weeks (5 weeks of age) and 6 months of treatment. FIG. 19A shows thecross-sections from tibialis anterior (TA) muscle stained withhematoxylin and eosin (HE) and NADH-TR, and by immunofluorescence withantibodies against DHPRla and dysferlin. The data in FIG. 2B depicts themean diameter of muscle fibers from TA and biceps brachii muscles frommice injected with either saline or rAAV-Mtm1 after 2 weeks (left graph)and 6 months of treatment (right graph).

FIG. 3A-3C demonstrate that gene replacement therapy with rAAV8-Mtm1improves strength, activity and long-term survival in myotubularindeficient mice. Whole-body spontaneous mobility of normal (WT+saline),mutant (KO+saline) and AAV-treated mutant (KO+AAV-Mtm1) mice 2 weeks (5weeks of age) and 6 months after PBS or vector injection was measured.The distance covered over the 90-min test was assessed using an openfield actimeter. FIG. 3A demonstrates that treatment increases distancetraveled. FIG. 3B depicts the escape test measurements in the 5 groupsof mice, again demonstrating that treatment increases function. FIG. 3Cdemonstrates the specific tetanic force of isolated EDL muscles from KOmice injected at an early and late stage of the disease 6 months aftervector delivery compared to saline-injected KO and WT littermates,showing again increased function in treated animals.

FIGS. 9A-9C demonstrate that systemic gene replacement therapyameliorates pathological hallmarks of myotubular myopathy in skeletalmuscles. Constitutive Mtm1 knockout mice (KO-Mtm1) at 3 weeks of agereceived a single intravenous injection of rAAV-Mtm1 as describedelsewhere herein. Saline injected KO-Mtm1 and WT mice served ascontrols. Distribution of myofiber diameters of tibialis anterior (TA,FIG. 9A, upper panels) and biceps brachii (BI, FIG. 9B lower panels)muscles at 2 weeks (5 weeks of age) and 6 months post-injection in thetwo group of animals (3 weeks and 5 weeks of age at injection), showingthat treatment restored fiber diameter to wild type distributions. Thepercentage of myofibers with internal nuclei was quantified in tibialisanterior muscle and biceps brachii of mice from the various treatmentconditions (FIG. 9B)

Systemic Administration of MTM1

The data presented herein document long-lasting benefits of a singlesystemic administration of rAAV-mediated myotubularin gene replacementtherapy in mouse models of XLMTM, a fatal congenital myopathy. Use ofthe muscle-specific desmin promoter to drive transgene expression wasvalidated by equivalent results in whole-body and muscle-restrictedknockout mice. Treatment with rAAV-Mtm1 reversed defects in skeletalmuscles throughout the body, and promoted survival and normal growth. Asassessed by histology, muscle strength, and global motor activity, genetherapy restored a nearly normal phenotype in myotubularin-deficientmice for at least one year.

Protein expression in response to varying amounts of rAAV-Mtm1 vectorassessed several months after injection indicated that myotubularinproduction was gene dose-dependent. At a higher vector level, the amountof myotubularin in treated KO-Mtm1 mice exceeded that in WT at 2 weekspost-injection, and by 6 months was more than 7-fold above normal inmuscles throughout the body. This potent response could be a potentialconcern because elevated expression of the homologous lipid phosphatase,myotubularin MTM-1 in Caenorhabditis elegans interferes withphagocytosis of apoptotic cells. Failure to remove cell corpses mayexacerbate autoimmunity and other disease processes. In addition, it wasfound previously that elevated levels of myotubularin in mouse myofiberscan lead to the accumulation of internal membrane saccules. However, inthe present study such pathological features were not evident.Furthermore, at a 6-fold lower rAAV-Mtm1 vector dose, the quantity ofmyotubularin protein in skeletal muscles of mKO-Mtm1 mice 12 monthspost-injection was similar to WT, and the muscular structure andfunction of treated animals appeared nearly normal. The thresholdmyotubularin content required for therapeutic benefit may beconsiderably lower. For example, mice engineered to have only tracemyotubularin activity, due to a missense mutation that also interfereswith RNA splicing, survive 8 times longer than absolutemyotubularin-knockout mutants. These findings suggest gene dose can beadjusted to express sufficient myotubularin enzymatic activity forefficacy, while avoiding potential side effects of supra-physiologicalproduction.

Local rAAV-mediated myotubularin gene therapy to a limb muscle of youngXLMTM dogs yielded conspicuous improvements in muscle gross morphology,myofiber size, and subcellular architecture compared to untreated limbs.Intravenous infusion with rAAV-MTM1 resulted in systemic effectsdemonstrated by robust improvement in histopathology, sarcotubularorganization and normalization of muscular strength. Remarkably, infusedXLMTM dogs remained robust with improved survival beyond 18weeks-of-age, with the first infused dog surviving for more than a year.Although pretreatment muscle biopsies were not obtained from these dogs,it is noted that skeletal muscles of myotubularin-deficient mice showsignificant sarcotubular disorganization by two weeks-of-age. Thus, ifsimilar disorganization is evident in the dog muscles during earlymaturation, these findings suggest that delivery of an MTM1 vector canreverse established pathological changes in mutant muscle and restorenormal structure. From a clinical perspective, the most importantobservation is the extraordinary and rapid functional improvementfollowing local or intravenous regional infusion of AAV8-MTM1 genetherapy. Untreated limb muscles of affected male dogs becameprogressively weaker than those of control littermates. By contrast, asmeasured in isometric and eccentric contraction tests, the strength ofvector-injected limbs gained significantly by 4 weeks and reached 70-80%of normal at 6 weeks. This finding holds true for intramuscular orintravenous administration of rAAV8-MTM1. In the present study, regionalintravenous infusion of vector suspended in saline under pressureagainst a tourniquet resulted in significant “leak” above the level ofthe tourniquet with vector transduction of non injected contralaterallimb muscles. The findings presented herein are in contrast to the lackof vector transduction of contralateral limbs or organs in a caninemodel of hemophilia following regional limb perfusion with AAV2 atcomparable doses. Reasons for these differences are not clear. Althoughblood flow was monitored using ultrasound above and below thetourniquet, the possibility of collateral circulation or of tourniquetslippage during the 10-minute infusion cannot be excluded.

The detailed mechanism by which myotubularin gene therapy corrects themuscle defects of mutant mice and dogs is not yet known. Importantfunctions controlled by members of the myotubularin phosphoinositide3-phospatase family include: endosomal and membrane trafficking andremodeling; surface localization of receptors and integrins;cytoskeletal dynamics; ion channel activity; and cell survival orapoptosis. Ablation of myotubularin in zebrafish or mice elevates PI(3)Pin muscle. This, in turn, inhibits phosphorylation of theserine/threonine kinase AKT, a key signal transduction enzyme regulatedby phosphoinositide second messengers. The precise pathway(s) by whichthis aberrant signaling leads to muscle cell failure in XLMTM remainsconjectural. However, expression profiling supports histologicalobservations pointing to remodeling of matrix, membrane, andcytoskeletal architecture as factors responsible for disorganization oforganelles and muscular hypotrophy.

A cellular feature closely associated with the muscular dysfunction inXLMTM is the disorganization of T-tubules. These specialized membranousstructures play an essential role in excitation-contraction coupling, bywhich nerve signals received by skeletal muscles are transmitted overthe sarcolemma as an action potential (AP). The AP is sensed by thedihydropyridine receptor in the T-tubule membrane which initiatesrelease of calcium via the ryanodine receptor from the sarcoplasmicreticulum. The increased cytrosolic calcium concentration activates theactin/myosin. Restoration of normal T-tubule architecture correlateswith the potent effect of myotubularin gene therapy on muscle strengthin murine and canine mutants.

Whatever the mechanistic basis, the beneficial results of gene therapyin the animal models encourage future steps towards clinicaltranslation. The data presented herein highlight the sustainedexpression of myotubularin in muscles of mutant small- and large-animalmodels treated with a single dose of recombinant adeno-associated virus.Genomes of rAAV vectors persist in transduced cells mainly asintranuclear concatameric episomes. This mode facilitates long-termcorrection of somatic cells, especially those that do not undergo rapidturnover. The rate of skeletal myofiber replacement in XLMTM patients isnot known. However, in contrast to Duchenne muscular dystrophy (DMD),the low degree of tissue inflammation associated with XLMTM invitesspeculation that muscle turnover is comparable to that in unaffectedindividuals. Normal skeletal myocytes are long-lived; birth dating ofhuman muscle cells, based on ¹⁴C levels in people who lived through theera of atmospheric nuclear testing, gave an average age of 15.1 years.

Immune responses to rAAV viral capsids or to the transgene product canpersist for years. If immune reactions appear problematic, theypotentially may be controlled by choice of administration route or bytreatments to induce immune tolerance. In the XLMTM dog, adverse immuneresponses to either the vector (AAV8) or the transgene (MTM1 protein)were not observed in the present study. Interestingly, long-termexpression of the MTM1 transgene was observed in the absence ofdetectable humoral or cellular immune responses against the restoredprotein, demonstrating in this animal model the lack of transgene-drivenimmune response.

It is anticipated that the clinical development of myotubularin genereplacement therapy will begin with local or loco-regional delivery.Targeting of wrist and hand muscles to enable control of a wheelchairand communication devices might improve quality of life for XLMTMpatients. Furthermore, most patients are ventilator-dependent andrequire continuous management. Targeted injection of the diaphragm couldrelieve this burden and potentially decrease mortality.

The sustained benefit of gene therapy in myotubularin-knockout micesuggests that systemic treatment of human patients eventually will befeasible. It is project that this could be carried out shortly afterbirth to decrease the risk of immune reaction. Molecular assays areavailable to rapidly identify MTM1 mutations in symptomatic newborns orprenatally. Systemic vector delivery in older individuals also appearsplausible, as evidenced by a recent study using rAAV8-mediated genetransfer in hemophilia B. Overall, the positive safety record of rAAVclinical trials, in which over 300 human subjects have enrolled sincethe mid-1990s, together with the completion of a Phase I clinical trialin DMD using an improved vector for gene therapy of muscle diseases,stimulate optimism for successful translation in XLMTM.

Example 2 Effects of Systemic AAV-MTM1 on Muscles of Respiration

The diaphragm is a parachute-shaped skeletal muscle that is the primarymuscle used in respiratory inspiration. The diaphragm extends across thebottom of the rib cage, separating the thoracic cavity from theabdominal cavity. During inhalation, the diaphragm contracts, reducingthoracic pressure and volume and causing air to be pulled into thelungs. During exhalation, the diaphragm relaxes and elastic recoil ofthe lungs occurs. The diaphragm is weakened in those with XLMTM, therebyleading to respiratory dysfunction. Given the location of the diaphragm,it is difficult, invasive, and often dangerous to provide local deliveryof therapeutics through intramuscular delivery to the diaphragm. It isdescribed herein, that system delivery of MTM1 surprisingly increasesthe strength and function of the diaphragm in subjects with MTM1deficiency.

The studies presented herein measure diaphragm strength usingRespiratory Impedance Plethysmography (RIP). Canine subjects were fittedwith a jacket with wired bands placed at the thorax and abdomen (emkaTechnology). Changes in low impedance current passed through the bandsare interpreted as changes in volume. (FIGS. 27A-27B). Anesthetized dogswere challenged with the respiratory stimulant, doxapram chloride.Doxapram acts centrally to stimulate respiration and thus allows for theassessment of the at-work diaphragm.

FIG. 28 depicts the change in percent abdominal contribution in responseto doxapram dose, as compared to baseline in dogs at 16 weeks of age.The data depicted in FIG. 28 demonstrates that systemic treatment withAAV8-MTM1 in XLMTM dogs improves the movement of muscle diaphragm suchthat the change in abdominal contribution is not different from wildtype.

In other experiments, the tidal breathing flow-volume loop (TBFVL) wasused to diagnose respiratory dysfunction. TBFVLs were developed toassess respiratory function in infants, where maximal voluntary effortis difficult. It has then adapted for use in cats and dogs. In normaldogs, the loop is D-shaped. TBFVLs are derived from pneumotach measuresof airflows. A pneumotach is used to measure airflow afteradministration of 1.0 mg/kg doxapram. As shown in FIG. 29, the volume isdetermined by finding the area under the curve of flow over time.Measures were then adjusted for weight and averaged.

As shown in FIG. 30, prior to treatment, the TBFVL for XLMTM dogs ismuch smaller compared to normal, wild type dogs. However, 8 weeks aftertreatment, TBFLV in treated dogs reveal much improved function. Becausequantitative analysis of TBFLV is difficult, peak inspiratory flow(PIF), was selected as a primary measure for quantifying respiratoryfunction (FIG. 31). Shown in FIG. 32 is PIF measured at 8 week and 16weeks of age in XLMTM dogs and their normal wild type littermates. Intreated XLMTM dogs, measures are taken just prior to AAV8-MTM1 genetherapy at 8 weeks of age; 16 week measures are two monthspost-treatment. In untreated XLMTM dogs, 16 weeks marks the terminal endpoint. Significance was determined by ANOVA with Bonferroni posttest(P<0.0001), with untreated XLMTM PIF being significantly lower than bothwild type and treated dogs. (N_(WT) at 8 weeks=3, N_(WT) at 16 weeks=⁵,N_(XLMTM) at 8 weeks=4, N_(Untreated) XLMTM at 16 weeks=4, N_(Treated)XLM at 16 weeks=3).

The data presented herein demonstrate that AAV8-MTM1 delivered by highpressure limb infusion results in improved respiratory muscle functionin XLMTM dogs.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed:
 1. A method of treating an X-linked myotubular myopathy(XLMTM) in a mammal in need thereof, the method comprising administeringto the mammal a composition that increases the expression ofmyotubularin in a muscle of the mammal, wherein the compositioncomprises a viral vector comprising a nucleic acid sequence encoding anMyotubularin 1 (MTM1) gene operably linked to a muscle specific promoterand wherein the muscle of the mammal administered the compositionexhibits an increase in MTM1 expression for a period selected from thegroup consisting of up to 3 months, up to 6 months and up to 1 year, ascompared with the muscle of a mammal not administered the composition.2. The method of claim 1, wherein the viral vector is selected from thegroup consisting of a lentiviral vector, retroviral vector, adenoviralvector, and adeno-associated viral (AAV) vector.
 3. The method of claim1, wherein the administration route comprises at least one selected fromthe group consisting of intravenous and intra-arterial.
 4. The method ofclaim 1, wherein the muscle of the mammal administered the compositionexhibits a sustained increase in strength for up to 3 months, ascompared with the muscle of a mammal not administered the composition.5. The method of claim 1, wherein the mammal administered thecomposition has a longer survival rate than a mammal not administeredthe composition.
 6. The method of claim 1, wherein the function of thediaphragm of the mammal administered the composition is improved, ascompared with the diaphragm of a mammal in the absence of administrationof the composition.
 7. The method of claim 1, wherein the administrationis selected from the group consisting of a single administration and atleast two administrations.
 8. A method of prolonging the survival of amammal with an X-linked myotubular myopathy (XLMTM), the methodcomprising administering to the mammal a composition that increases theexpression of myotubularin in a muscle of the mammal, wherein thecomposition comprises a viral vector comprising a nucleic acid sequenceencoding an Myotubularin 1 (MTM1) gene operably linked to a musclespecific promoter and wherein the muscle of the mammal administered thecomposition exhibits an increase in MTM1 expression for a periodselected from the group consisting of up to 3 months, up to 6 months andup to 1 year, as compared with the muscle of a mammal not administeredthe composition.
 9. The method of claim 8, wherein the viral vector isselected from the group consisting of a lentiviral vector, retroviralvector, adenoviral vector, and adeno-associated viral (AAV) vector. 10.The method of claim 8, wherein the administration route comprises atleast one selected from the group consisting of intravenous andintra-arterial.
 11. The method of claim 8, wherein the muscle of themammal administered the composition exhibits a sustained increase instrength for up to 3 months, as compared with the muscle of a mammal notadministered the composition.
 12. The method of claim 8, wherein thefunction of the diaphragm of the mammal administered the composition isimproved, as compared with the diaphragm of a mammal in the absence ofadministration of the composition.
 13. The method of claim 8, whereinthe administration is selected from the group consisting of a singleadministration and at least two administrations.
 14. A compositioncomprising a viral vector comprising a nucleic acid sequence comprisingan Myotubularin 1 (MTM1) gene operably linked to a muscle specificpromoter.
 15. The composition of claim 14, wherein the viral vector isselected from the group consisting of a viral vector, lentiviral vector,retroviral vector, adenoviral vector, and adeno-associated viral (AAV)vector.
 16. The composition of claim 14, wherein the muscle specificpromoter is a desmin promoter.
 17. A method of restoring normal musclefunction in a mammal with an X-linked myotubular myopathy (XLMTM), themethod comprising administering to the mammal a composition thatincreases the expression of myotubularin in a muscle of the mammal,wherein the composition comprises a viral vector comprising a nucleicacid sequence encoding an Myotubularin 1 (MTM1) gene operably linked toa muscle specific promoter and wherein the muscle of the mammaladministered the composition exhibits an increase in MTM1 expression toachieve a level similar to the MTM1 expression in the muscle of ahealthy mammal.
 18. The method of claim 17, wherein the viral vector isselected from the group consisting of a lentiviral vector, retroviralvector, adenoviral vector, and adeno-associated viral (AAV) vector. 19.The method of claim 17, wherein the administration route comprises atleast one selected from the group consisting of intravenous andintra-arterial.
 20. The method of claim 17, wherein the muscle of themammal administered the composition exhibits sustained increase instrength to reach a level similar to the muscle of a healthy mammal. 21.The method of claim 17, wherein the function of the diaphragm of themammal administered the composition exhibits sustained improvement toreach a level similar to the function of the diaphragm of a healthymammal.
 22. The method of claim 17, wherein the administration isselected from the group consisting of a single administration and atleast two administrations.
 23. A method of treating an X-linkedmyotubular myopathy (XLMTM) in a mammal with an early onset of thepathology, the method comprising administering to the mammal acomposition that increases the expression of myotubularin in a muscle ofthe mammal, wherein the composition comprises a viral vector comprisinga nucleic acid sequence encoding an Myotubularin 1 (MTM1) gene operablylinked to a muscle specific promoter.
 24. The method of claim 23,wherein the viral vector is selected from the group consisting of alentiviral vector, retroviral vector, adenoviral vector, andadeno-associated viral (AAV) vector.
 25. The method of claim 23, whereinthe administration route comprises at least one selected from the groupconsisting of intravenous and intra-arterial.
 26. The method of claim23, wherein the administration is selected from the group consisting ofa single administration and at least two administrations.
 27. The methodof claim 2, wherein the AAV vector comprises a serotype selected fromthe group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,and AAV9.
 28. The method of claim 9, wherein the AAV vector comprises aserotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, and AAV9.
 29. The composition of claim 15,wherein the AAV vector comprises a serotype selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.30. The method of claim 18, wherein the AAV vector comprises a serotypeselected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, and AAV9.
 31. The method of claim 24, wherein the AAVvector comprises a serotype selected from the group consisting of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.